Methods and compositions for eliciting an immune response against hepatitis b virus

ABSTRACT

The present invention relates to immunization of hypo-responsive groups of individuals. In particular, the present invention provides methods and compositions for eliciting a potent immune response to hepatitis B virus in individuals in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/475,548, filed Apr. 14, 2011, and U.S. Provisional Application No.61/559,054, filed Nov. 12, 2011, which are both hereby incorporated byreference herein in their entirety.

SUBMISSION OF SEQUENCE LISTING AS ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 377882005200SeqList.txt,date recorded: Apr. 13, 2012, size: 1 KB).

FIELD

The present invention relates to immunization of hypo-responsive groupsof individuals. In particular, the present invention provides methodsand compositions for eliciting a potent immune response to hepatitis Bvirus in individuals in need thereof.

BACKGROUND

Hepatitis B virus (HBV) is one of several viruses known to cause liverdisease (e.g., chronic active hepatitis, cirrhosis, liver failure andhepatocellular carcinoma) in humans. HBV is spread through percutaneousor mucosal contact with infected body fluids. According to the WorldHealth Organization (WHO), nearly two billion people are infected withHBV, which causes over 600,000 fatalities each year (WHO, Fact Sheet No.204, 2009).

The HBV virion is composed of a core antigen (HBcAg), which encapsulatesviral DNA, and a surface antigen (HBsAg), which is located on the viralouter membrane. HBsAg, previously known as the Australian antigen, iscomposed of three glycoproteins having a shared carboxy terminalsequence: S (S only); M (pre-S2 and S); and L (pre-S1, pre-S2 and S).HBsAg self-associates to form 22 nm particles that are released frominfected hepatocytes.

All HBV vaccines approved for use in humans are based on noninfectiousHBsAg particles. In the United States, the current HBV vaccines arerecombinant subunit vaccines produced in yeast (e.g., RECOMBIVAX HB®hepatitis B vaccine marketed by Merck & Co.; and ENGERIX-B® hepatitis Bvaccine marketed by GlaxoSmithKline). These HBV vaccines are formulatedas HBsAg adsorbed to alum.

Use of the current vaccines is hindered by the typical lengthyadministration regimen (e.g., generally three or four doses over six totwelve months). In addition, only 10-20% of adult vaccine recipientsmount a seroprotective immune response within one month of receiving afirst dose of a HBV vaccine (Andre and Sarary, Post Grad Med J, 63:169-178, 1987; and Keating and Noble, Drugs 2003, 63:1021-1051, 2003).This delay in the generation of a protective antibody response is ofparticular importance for individuals at high risk of HBV infection(e.g., health care workers, emergency first responders, or individualsin high-risk behavior groups). Further, compliance in returning forthree or four injections over six to twelve months can be poor.

Another serious shortcoming of the current HBV vaccines is the highlevel of hypo- or non-responders (30-60% hypo- or nonresponders afterthe recommended regimen) among some groups, such as those over 40 yearsof age (Denis et al., J Infect Dis, 149:1019, 1984; Averoff et al., Am JPrev Med, 15:1-8, 1998; and Treadwell et al., Am J Med, 95:584-588,1993), and subjects with renal failure or diabetes (Weber et al., JAMA,254:3187-3189, 1985). Thus, development of a more potent HBV vaccinewith more rapid induction of protective immunity, and improved responseamong hypo-responder populations is of major importance in reducing HBVinfection.

SUMMARY

The present invention relates to immunization of hypo-responsive groupsof individuals. In particular, the present invention provides methodsand compositions for eliciting a potent immune response to hepatitis Bvirus in individuals in need thereof.

Specifically, the present disclosure provides methods for eliciting animmune response against hepatitis B virus (HBV) in a human subjecthaving a glucose metabolism disorder, comprising: administering to ahuman subject a first and a second dose of an immunogenic composition ontwo separate occasions (e.g., first dose at week 0 and a second dose atweek 4), wherein the immunogenic composition comprises a hepatitis Bsurface antigen (HBsAg) and an immunostimulatory sequence (ISS) of from8 to 50 nucleotides in length comprising an unmethylatedcytosine-phosphate-guanosine (CpG) motif, wherein the subject has aglucose metabolism disorder selected from the group consisting of type Idiabetes, type II diabetes, and pre-diabetes at the onset of theadministering; and wherein the HBsAg and the ISS are present in theimmunogenic composition in amounts effective to elicit an immuneresponse in the subject by two months after the second dose. In somepreferred embodiments, the present disclosure provides methods foreliciting a seroprotective immune response against HBV in a diabetichuman subject, comprising: administering to a diabetic human subject afirst and a second dose of an immunogenic composition on two separateoccasions (e.g., first dose at week 0 and a second dose at week 4),wherein the immunogenic composition comprises a hepatitis B surfaceantigen (HBsAg) and an immunostimulatory sequence (ISS) of from 8 to 50nucleotides in length comprising an unmethylatedcytosine-phosphate-guanosine (CpG) motif, wherein the HBsAg and the ISSare present in the immunogenic composition in amounts effective toelicit a seroprotective immune response comprising an anti-HBsAgconcentration of at least 10 mIU/ml in the subject by two months afterthe second dose. In some embodiments, the methods further compriseadministering a subunit vaccine comprising HBsAg adsorbed to alum as asubsequent dose (e.g., a booster at least 6 months after the first dose,preferably at least 1 year, 5 years or 10 years after the first dose).In some embodiments, the ISS comprises the nucleotide sequence5′-TCG-3′. In some embodiments, the ISS comprises the nucleotidesequence ‘5-CGTTCG-3’ or ‘5-AACGTTCG-3’. In some embodiments, the ISScomprises the nucleotide sequence of SEQ ID NO:1. In other embodiments,the ISS comprises the nucleotide sequence of SEQ ID NO:2. In somepreferred embodiments, the ISS comprises a phosphate backbonemodification. In some preferred embodiments, the ISS comprises aphosphorothioate backbone modification. In some embodiments, the ISS issingle-stranded. In some embodiments, the ISS is double-stranded. Insome preferred embodiments, the ISS is 1018 ISS. In some embodiments,the immunogenic composition comprises 20 μg or less (e.g., 0.2-20 μg) ofthe HBsAg. In some embodiments, the immunogenic composition comprises3000 μg or less (e.g., 30-3000 μg) of the ISS. In some preferredembodiments, the immunogenic composition comprises about 20 μg of theHBsAg and about 3000 μg of the ISS. In some embodiments, the immunogeniccomposition further comprises a buffer. In some embodiments, the buffercomprises sodium phosphate and sodium chloride. In some preferredembodiments, the immunogenic composition is buffered from pH 6.5 to 7.5,or to about pH 7.0. In some embodiments, the immunogenic compositionfurther comprises a surfactant. In a subset of these embodiments, thesurfactant comprises polysorbate. In some embodiments, the immunogeniccomposition further comprises an additional adjuvant. In a subset ofthese embodiments, the additional adjuvant is comprises alum. In somepreferred embodiments, the HBsAg is a recombinant HBsAg produced inyeast. In some preferred embodiments, the immune response is aseroprotective antibody response comprising an anti-HBsAg response of atleast 10 mIU/mL by two months after the second dose. In a subset ofthese embodiments, the anti-HBsAg response is at least 15, 20, or 25mIU/mL by two months after the second dose. In some embodiments, theimmune response is a seroprotective antibody response comprising ananti-HBsAg response of at least 10 mIU/mL by six months after the seconddose. In a subset of these embodiments, the anti-HBsAg response is atleast 20, 30, 40 or 50 mIU/mL by six months after the second dose. Insome preferred embodiments, the seroprotective antibody response isstatistically greater than that elicited by administration of a controlimmunogenic composition lacking the ISS. In some preferred embodiments,the subject has type II diabetes. In some embodiments, the subject istaking one or both of an oral hypoglycemic and insulin, at the onset ofthe administering. In some embodiments, the oral hypoglycemic comprisesone or more of the group consisting of a biguanide, a sulfonylurea, anonsulfonylurea secretagogue, an alpha glucosidase inhibitor, and athiazolidinedione. In some embodiments, the biguanide is metformin. Insome embodiments, the insulin is recombinant human insulin or an analogthereof. In some embodiments, the human subject is a diabetic humansubject with chronic kidney disease. In some preferred embodiments, thesubject has a body mass index of greater than 25 kg/m² (overweight). Inother preferred embodiments, the subject has a body mass index ofgreater than 30 kg/m² (obese), or a body mass index of greater than 40kg/m² (morbidly obese). In some embodiments, the subject is a residentof a nursing home or an assisted living facility. In other embodiments,the subject is a resident of a correctional facility. In someembodiments, the subject is over 40 years of age or 40 years of age orolder. In a subset of these embodiments, the subject is from 41 to 60 or61 to 80 years of age. In some preferred embodiments, the HBsAgcomprises the S antigen. In other preferred embodiments, the HBsAgfurther comprises one or both of the pre-S2 antigen, and the pre-S1antigen. In some embodiments, the HBsAg antigen is purified from plasmaof an HBV-infected subject. In other embodiments, the HBsAg antigen is arecombinant HBsAg produced in mammalian cells in vitro. In furtherembodiments, the present disclosure provides methods for eliciting animmune response against hepatitis B virus (HBV) in a human subjecthaving a glucose metabolism disorder, comprising: administering to ahuman subject an effective amount of an immunogenic composition, whereinthe immunogenic composition comprises a hepatitis B surface antigen(HBsAg), and an immunostimulatory sequence (ISS) of from 8 to 50nucleotides in length comprising an unmethylatedcytosine-phosphate-guanosine (CpG) motif, and wherein the subject has aglucose metabolism disorder selected from the group consisting of type Idiabetes, type II diabetes, and pre-diabetes at the onset of theadministering. In some embodiments, the immunogenic composition isadministered on two separate occasions as a first dose and a seconddose, and the effective amount is sufficient to elicit an immuneresponse in the subject after the second dose. Also provided are kitsfor eliciting an immune response against hepatitis B virus (HBV) in ahuman subject having a glucose metabolism disorder, comprising: animmunogenic composition comprising a hepatitis B surface antigen(HBsAg), and an immunostimulatory sequence (ISS) of from 8 to 50nucleotides in length comprising an unmethylatedcytosine-phosphate-guanosine (CpG) motif; and instructions foradministering to a human subject an effective amount of the immunogeniccomposition, wherein the subject has a glucose metabolism disorderselected from the group consisting of type I diabetes, type II diabetes,and pre-diabetes.

Additionally, the present disclosure provides immunogenic compositionscomprising an immunostimulatory sequence (ISS) and a hepatitis B surfaceantigen (HBsAg) for use in eliciting an immune response againsthepatitis B virus (HBV) in a human subject when administered as a firstdose and a second dose on two separate occasions (e.g., first dose atweek 0 and a second dose at week 4), wherein the ISS is from 8 to 50nucleotides in length comprising an unmethylatedcytosine-phosphate-guanosine (CpG) motif, wherein the ISS and the HBsAgare present in the immunogenic composition in amounts effective toelicit an immune response in the subject by two months after the seconddose, and wherein the subject has a glucose metabolism disorder selectedfrom the group consisting of type I diabetes, type II diabetes, andpre-diabetes at the onset of the administering. Also provided by thepresent disclosure are immunogenic compositions comprising aimmunostimulatory sequence (ISS) and a hepatitis B surface antigen(HBsAg) for use in preventing a human subject from being infected with ahepatitis B virus (HBV) when administered as a first dose and a seconddose on two separate occasions, wherein the ISS is from 8 to 50nucleotides in length comprising an unmethylatedcytosine-phosphate-guanosine (CpG) motif, wherein the ISS and the HBsAgare present in the immunogenic composition in amounts effective toprevent the subject from becoming infected with HBV by two months afterthe second dose, and wherein the subject has a glucose metabolismdisorder selected from the group consisting of type I diabetes, type IIdiabetes, and pre-diabetes at the onset of the administering. In someembodiments, the ISS comprises the nucleotide sequence 5′-TCG-3′. Insome embodiments, the ISS comprises the nucleotide sequence ‘5-CGTTCG-3’or ‘5-AACGTTCG-3’. In some embodiments, the ISS comprises the nucleotidesequence of SEQ ID NO:1. In other embodiments, the ISS comprises thenucleotide sequence of SEQ ID NO:2. In some preferred embodiments, theISS comprises a phosphate backbone modification. In some preferredembodiments, the ISS comprises a phosphorothioate backbone modification.In some embodiments, the ISS is single-stranded. In some embodiments,the ISS is double-stranded. In some preferred embodiments, the ISS is1018 ISS. In some embodiments, the immunogenic composition comprises 20μg or less (e.g., 0.2-20 μg) of the HBsAg. In some embodiments, theimmunogenic composition comprises 3000 μg or less (e.g., 30-3000 μg) ofthe ISS. In some preferred embodiments, the immunogenic compositioncomprises about 20 μg of the HBsAg and about 3000 μg of the ISS. In someembodiments, the immunogenic composition further comprises a buffer. Insome embodiments, the buffer comprises sodium phosphate and sodiumchloride. In some preferred embodiments, the immunogenic composition isbuffered from pH 6.5 to 7.5, or to about pH 7.0. In some embodiments,the immunogenic composition further comprises a surfactant. In a subsetof these embodiments, the surfactant comprises polysorbate. In someembodiments, the immunogenic composition further comprises an additionaladjuvant. In a subset of these embodiments, the additional adjuvant iscomprises alum. In some preferred embodiments, the HBsAg is arecombinant HBsAg produced in yeast. In some preferred embodiments, theimmune response is a seroprotective antibody response comprising ananti-HBsAg response of at least 10 mIU/mL by two months after the seconddose. In a subset of these embodiments, the anti-HBsAg response is atleast 15, 20, or 25 mIU/mL by two months after the second dose. In someembodiments, the immune response is a seroprotective antibody responsecomprising an anti-HBsAg response of at least 10 mIU/mL by six monthsafter the second dose. In a subset of these embodiments, the anti-HBsAgresponse is at least 20, 30, 40 or 50 mIU/mL by six months after thesecond dose. In some preferred embodiments, the seroprotective antibodyresponse is statistically greater than that elicited by administrationof a control immunogenic composition lacking the ISS. In some preferredembodiments, the subject has type II diabetes. In some embodiments, thesubject is taking one or both of an oral hypoglycemic and insulin, atthe onset of the administering. In some embodiments, the oralhypoglycemic comprises one or more of the group consisting of abiguanide, a sulfonylurea, a nonsulfonylurea secretagogue, an alphaglucosidase inhibitor, and a thiazolidinedione. In some embodiments, thebiguanide is metformin. In some embodiments, the insulin is recombinanthuman insulin or an analog thereof. In some embodiments, the humansubject is a diabetic human subject with chronic kidney disease. In somepreferred embodiments, the subject has a body mass index of greater than25 kg/m² (overweight). In other preferred embodiments, the subject has abody mass index of greater than 30 kg/m² (obese), or a body mass indexof greater than 40 kg/m² (morbidly obese). In some embodiments, thesubject is a resident of a nursing home or an assisted living facility.In other embodiments, the subject is a resident of a correctionalfacility. In some embodiments, the subject is over 40 years of age or 40years of age or older. In a subset of these embodiments, the subject isfrom 41 to 60 or 61 to 80 years of age. In some preferred embodiments,the HBsAg comprises the S antigen. In other preferred embodiments, theHBsAg further comprises one or both of the pre-S2 antigen, and thepre-S1 antigen. In some embodiments, the HBsAg antigen is purified fromplasma of an HBV-infected subject. In other embodiments, the HBsAgantigen is a recombinant HBsAg produced in mammalian cells in vitro.

Moreover, the present disclosure provides immunogenic compositionscomprising an immunostimulatory sequence (ISS) and a hepatitis B surfaceantigen (HBsAg) for use in preparing a medicament to elicit an immuneresponse against hepatitis B virus (HBV) in a human subject whenadministered as a first dose and a second dose on two separate occasions(e.g., first dose at week 0 and a second dose at week 4), wherein theISS is from 8 to 50 nucleotides in length comprising an unmethylatedcytosine-phosphate-guanosine (CpG) motif, wherein the ISS and the HBsAgare present in the immunogenic composition in amounts effective toelicit an immune response in the subject by two months after the seconddose, and wherein the subject has a glucose metabolism disorder selectedfrom the group consisting of type I diabetes, type II diabetes, andpre-diabetes at the onset of the administering. Also provided by thepresent disclosure are immunogenic compositions comprising animmunostimulatory sequence (ISS) and a hepatitis B surface antigen(HBsAg) for use in preparing a medicament for preventing a human subjectfrom being infected with a hepatitis B virus (HBV) when administered asa first dose and a second dose on two separate occasions, wherein theISS is from 8 to 50 nucleotides in length comprising an unmethylatedcytosine-phosphate-guanosine (CpG) motif, wherein the ISS and the HBsAgare present in the immunogenic composition in amounts effective toprevent the subject from becoming infected with HBV by two months afterthe second dose, and wherein the subject has a glucose metabolismdisorder selected from the group consisting of type I diabetes, type IIdiabetes, and pre-diabetes at the onset of the administering. In someembodiments, the ISS comprises the nucleotide sequence 5′-TCG-3′. Insome embodiments, the ISS comprises the nucleotide sequence ‘5-CGTTCG-3’or ‘5-AACGTTCG-3’. In some embodiments, the ISS comprises the nucleotidesequence of SEQ ID NO:1. In other embodiments, the ISS comprises thenucleotide sequence of SEQ ID NO:2. In some preferred embodiments, theISS comprises a phosphate backbone modification. In some preferredembodiments, the ISS comprises a phosphorothioate backbone modification.In some embodiments, the ISS is single-stranded. In some embodiments,the ISS is double-stranded. In some preferred embodiments, the ISS is1018 ISS. In some embodiments, the immunogenic composition comprises 20μg or less (e.g., 0.2-20 μg) of the HBsAg. In some embodiments, theimmunogenic composition comprises 3000 μg or less (e.g., 30-3000 μg) ofthe ISS. In some preferred embodiments, the immunogenic compositioncomprises about 20 μg of the HBsAg and about 3000 μg of the ISS. In someembodiments, the immunogenic composition further comprises a buffer. Insome embodiments, the buffer comprises sodium phosphate and sodiumchloride. In some preferred embodiments, the immunogenic composition isbuffered from pH 6.5 to 7.5, or to about pH 7.0. In some embodiments,the immunogenic composition further comprises a surfactant. In a subsetof these embodiments, the surfactant comprises polysorbate. In someembodiments, the immunogenic composition further comprises an additionaladjuvant. In a subset of these embodiments, the additional adjuvant iscomprises alum. In some preferred embodiments, the HBsAg is arecombinant HBsAg produced in yeast. In some preferred embodiments, theimmune response is a seroprotective antibody response comprising ananti-HBsAg response of at least 10 mIU/mL by two months after the seconddose. In a subset of these embodiments, the anti-HBsAg response is atleast 15, 20, or 25 mIU/mL by two months after the second dose. In someembodiments, the immune response is a seroprotective antibody responsecomprising an anti-HBsAg response of at least 10 mIU/mL by six monthsafter the second dose. In a subset of these embodiments, the anti-HBsAgresponse is at least 20, 30, 40 or 50 mIU/mL by six months after thesecond dose. In some preferred embodiments, the seroprotective antibodyresponse is statistically greater than that elicited by administrationof a control immunogenic composition lacking the ISS. In some preferredembodiments, the subject has type II diabetes. In some embodiments, thesubject is taking one or both of an oral hypoglycemic and insulin, atthe onset of the administering. In some embodiments, the oralhypoglycemic comprises one or more of the group consisting of abiguanide, a sulfonylurea, a nonsulfonylurea secretagogue, an alphaglucosidase inhibitor, and a thiazolidinedione. In some embodiments, thebiguanide is metformin. In some embodiments, the insulin is recombinanthuman insulin or an analog thereof. In some embodiments, the humansubject is a diabetic human subject with chronic kidney disease. In somepreferred embodiments, the subject has a body mass index of greater than25 (overweight). In other preferred embodiments, the subject has a bodymass index of greater than 30 (obese), or a body mass index of greaterthan 40 (morbidly obese). In some embodiments, the subject is a residentof a nursing home or an assisted living facility. In other embodiments,the subject is a resident of a correctional facility. In someembodiments, the subject is over 40 years of age. In a subset of theseembodiments, the subject is from 41 to 60 or 61 to 80 years of age. Insome preferred embodiments, the HBsAg comprises the S antigen. In otherpreferred embodiments, the HBsAg further comprises one or both of thepre-S2 antigen, and the pre-S1 antigen. In some embodiments, the HBsAgantigen is purified from plasma of an HBV-infected subject. In otherembodiments, the HBsAg antigen is a recombinant HBsAg produced inmammalian cells in vitro.

The present disclosure further provides methods for eliciting a highrate of seroprotection against hepatitis B virus (HBV) in ahypo-responsive human population, comprising: administering to thehypo-responsive human population a first and a second dose of animmunogenic composition on two separate occasions, wherein theimmunogenic composition comprises a hepatitis B surface antigen (HBsAg)and an immunostimulatory sequence (ISS) of from 8 to 50 nucleotides inlength comprising an unmethylated cytosine-phosphate-guanosine (CpG)motif, and wherein the HBsAg and the ISS are present in the immunogeniccomposition in amounts effective to elicit a high rate of seroprotectionagainst HBV in the hypo-responsive population by two months after thesecond dose. In some embodiments, the HBsAg and the ISS are present inthe immunogenic composition in amounts effective to elicit a high rateof seroprotection against HBV in the hypo-responsive population by sixmonths after the second dose. In some embodiments, the high rate ofseroprotection comprises a seroprotection rate of least 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% or 95% of the hypo-responsivepopulation receiving the immunogenic composition. Also provided aremethods for eliciting a seroprotective immune response against hepatitisB virus (HBV) in a human subject who is a member of a hypo-responsivepopulation, comprising: administering to the human subject a first and asecond dose of an immunogenic composition on two separate occasions(e.g., first dose at week 0 and a second dose at week 4), wherein theimmunogenic composition comprises a hepatitis B surface antigen (HBsAg)and an immunostimulatory sequence (ISS) of from 8 to 50 nucleotides inlength comprising an unmethylated cytosine-phosphate-guanosine (CpG)motif, and wherein the HBsAg and the ISS are present in the immunogeniccomposition in amounts effective to elicit a seroprotective immuneresponse against HBV in the human subject by two months after the seconddose. In some embodiments, the HBsAg and the ISS are present in theimmunogenic composition in amounts effective to elicit a seroprotectiveimmune response against HBV in the human subject by six months after thesecond dose. In some embodiments, the seroprotective immune responsecomprises an anti-HBsAg concentration of at least 15, 20, 25, or 30mUI/mL In some embodiments, the hypo-responsive population is selectedfrom the group consisting of older adults, males, obese individuals,smokers, type I diabetics, type II diabetics, patients with chronickidney disease, patients with chronic liver disease, patients receivingimmunosuppressive therapy (transplant recipients), patients receivingchemotherapy (cancer patients), HIV-infected individuals, andHCV-infected individuals. In some embodiments, the hypo-responsivepopulation comprises one or more of older adults, males, obeseindividuals, smokers, type I diabetics, type II diabetics, patients withchronic kidney disease, patients with chronic liver disease, patientsreceiving immunosuppressive therapy (transplant recipients), patientsreceiving chemotherapy (cancer patients), HIV-infected individuals, andHCV-infected individuals. Further variations of these methods aredescribed in the preceding paragraphs.

Additionally, the present disclosure provides methods for eliciting ahigh level of seroprotection against hepatitis B virus (HBV) in ahypo-responsive human population according to an abbreviated vaccinationschedule, comprising: administering to the hypo-responsive humanpopulation an immunogenic composition according to an abbreviatedvaccination schedule, wherein the abbreviated vaccination schedulecomprises administering a first, second and third dose of theimmunogenic composition on three separate occasions, wherein theimmunogenic composition comprises a hepatitis B surface antigen (HBsAg)and an immunostimulatory sequence (ISS) of from 8 to 50 nucleotides inlength comprising an unmethylated cytosine-phosphate-guanosine (CpG)motif, wherein the HBsAg and the ISS are present in the immunogeniccomposition in amounts effective to elicit a high level ofseroprotection against HBV in the hypo-responsive population by onemonth after the third dose. In some embodiments, the high level ofseroprotection comprises a seroprotection rate of least 80%, 85%, 90%,91%, 92%, 93%, 94% or 95% of the hypo-responsive population receivingthe immunogenic composition. In some embodiments, the high level ofseroprotection comprises an anti-HBsAg geometric mean concentration ofat least 50, 100, 150, 200, 250, 300, 350, 400 or 450 mUI/mL. Alsoprovided are methods for eliciting a seroprotective immune responseagainst hepatitis B virus (HBV) in a human subject who is a member of ahypo-responsive population according to an abbreviated vaccinationschedule, comprising: administering to the human subject an immunogeniccomposition according to an abbreviated vaccination schedule, whereinthe abbreviated vaccination schedule comprises administering a first,second and third dose of the immunogenic composition on three separateoccasions (e.g., first dose at week 0, a second dose at week 4, and athird dose at week 24), wherein the immunogenic composition comprises ahepatitis B surface antigen (HBsAg), and an immunostimulatory sequence(ISS) of from 8 to 50 nucleotides in length comprising an unmethylatedcytosine-phosphate-guanosine (CpG) motif, wherein the HBsAg and the ISSare present in the immunogenic composition in amounts effective toelicit a seroprotective immune response comprising an anti-HBsAgconcentration of at least 10 mUI/ml in the human subject by one monthafter the third dose. In some embodiments, the seroprotective immuneresponse comprises an anti-HBsAg concentration of at least 15, 20, 25,or 30 mUI/mL by one month after the third dose. In some embodiments, theseroprotective immune response comprises an anti-HBsAg concentration ofat least 40, 50, 100, 150, 200, 250, 300, 350, 400 or 450 mUI/mL. Insome embodiments, the hypo-responsive population is selected from thegroup consisting of older adults, males, obese individuals, smokers,type I diabetics, type II diabetics, patients with chronic kidneydisease, patients with chronic liver disease, patients receivingimmunosuppressive therapy (transplant recipients), patients receivingchemotherapy (cancer patients), HIV-infected individuals, andHCV-infected individuals. In some embodiments, the hypo-responsivepopulation comprises one or more of older adults, males, obeseindividuals, smokers, diabetics (type I diabetics and type II diabetics)and patients with chronic kidney disease. In some embodiments, the humansubject has chronic kidney disease. In some embodiments, the humansubject has chronic kidney disease and diabetes. In some embodiments,the ISS comprises the nucleotide sequence 5′-TCG-3′. In someembodiments, the ISS comprises the nucleotide sequence ‘5-CGTTCG-3’ or‘5-AACGTTCG-3’. In some embodiments, the ISS comprises the nucleotidesequence of SEQ ID NO:1. In other embodiments, the ISS comprises thenucleotide sequence of SEQ ID NO:2. In some preferred embodiments, theISS comprises a phosphate backbone modification. In some preferredembodiments, the ISS comprises a phosphorothioate backbone modification.In some embodiments, the ISS is single-stranded. In some embodiments,the ISS is double-stranded. In some preferred embodiments, the ISS is1018 ISS. In some embodiments, the immunogenic composition comprises 20μg or less (e.g., 0.2-20 μg) of the HBsAg. In some embodiments, theimmunogenic composition comprises 3000 μg or less (e.g., 30-3000 μg) ofthe ISS. In some preferred embodiments, the immunogenic compositioncomprises about 20 μg of the HBsAg and about 3000 μg of the ISS. In someembodiments, the immunogenic composition further comprises a buffer. Insome embodiments, the buffer comprises sodium phosphate and sodiumchloride. In some preferred embodiments, the immunogenic composition isbuffered from pH 6.5 to 7.5, or to about pH 7.0. In some embodiments,the immunogenic composition further comprises a surfactant. In a subsetof these embodiments, the surfactant comprises polysorbate. In someembodiments, the immunogenic composition further comprises an additionaladjuvant. In a subset of these embodiments, the additional adjuvant iscomprises alum. In some preferred embodiments, the HBsAg is arecombinant HBsAg produced in yeast. In some preferred embodiments, theHBsAg comprises the S antigen. In other preferred embodiments, the HBsAgfurther comprises one or both of the pre-S2 antigen, and the pre-S1antigen. In some embodiments, the HBsAg antigen is purified from plasmaof an HBV-infected subject. In other embodiments, the HBsAg antigen is arecombinant HBsAg produced in mammalian cells in vitro. Furthervariations of these methods are described in the preceding paragraphs.

Moreover, the present disclosure provides methods for quickly elicitingan immune response against hepatitis B virus (HBV) in a hypo-responsivehuman population, comprising: administering to the hypo-responsive humanpopulation a first and a second dose of an immunogenic composition ontwo separate occasions, wherein the immunogenic composition comprises ahepatitis B surface antigen (HBsAg) and an immunostimulatory sequence(ISS) of from 8 to 50 nucleotides in length comprising an unmethylatedcytosine-phosphate-guanosine (CpG) motif, wherein the HBsAg and the ISSare present in the immunogenic composition in amounts effective toelicit an immune response comprising an anti-HBsAg geometric meanconcentration of at least 5 mUI/mL in the hypo-responsive population bytwo months after the second dose. In some embodiments, the anti-HBsAggeometric mean concentration is at least 7.5, 10, 15 or 20 mUI/mL. Alsoprovided are methods for quickly eliciting an immune response againsthepatitis B virus (HBV) in a human subject who is a member of ahypo-responsive population, comprising: administering to the humansubject a first and a second dose of an immunogenic composition on twoseparate occasions (e.g., first dose at week 0, and a second dose atweek 4), wherein the immunogenic composition comprises a hepatitis Bsurface antigen (HBsAg) and an immunostimulatory sequence (ISS) of from8 to 50 nucleotides in length comprising an unmethylatedcytosine-phosphate-guanosine (CpG) motif, wherein the HBsAg and the ISSare present in the immunogenic composition in amounts effective toelicit an immune response comprising an anti-HBsAg concentration of atleast 5 mUI/mL in the human subject by two months after the second dose.In some embodiments, the anti-HBsAg concentration is at least 7.5, 10,15 or 20 mUI/mL by two months after the second dose. In someembodiments, the hypo-responsive population is selected from the groupconsisting of older adults, males, obese individuals, smokers, type Idiabetics, type II diabetics, patients with chronic kidney disease,patients with chronic liver disease, patients receivingimmunosuppressive therapy (transplant recipients), patients receivingchemotherapy (cancer patients), HIV-infected individuals, andHCV-infected individuals. In some embodiments, the hypo-responsivepopulation comprises one or more of older adults, males, obeseindividuals, smokers, diabetics (type I diabetics and type IIdiabetics), and patients with chronic kidney disease. In someembodiments, the ISS comprises the nucleotide sequence 5′-TCG-3′. Insome embodiments, the ISS comprises the nucleotide sequence ‘5-CGTTCG-3’or ‘5-AACGTTCG-3’. In some embodiments, the ISS comprises the nucleotidesequence of SEQ ID NO:1. In other embodiments, the ISS comprises thenucleotide sequence of SEQ ID NO:2. In some preferred embodiments, theISS comprises a phosphate backbone modification. In some preferredembodiments, the ISS comprises a phosphorothioate backbone modification.In some embodiments, the ISS is single-stranded. In some embodiments,the ISS is double-stranded. In some preferred embodiments, the ISS is1018 ISS. In some embodiments, the immunogenic composition comprises 20μg or less (e.g., 0.2-20 μg) of the HBsAg. In some embodiments, theimmunogenic composition comprises 3000 μg or less (e.g., 30-3000 μg) ofthe ISS. In some preferred embodiments, the immunogenic compositioncomprises about 20 μg of the HBsAg and about 3000 μg of the ISS. In someembodiments, the immunogenic composition further comprises a buffer. Insome embodiments, the buffer comprises sodium phosphate and sodiumchloride. In some preferred embodiments, the immunogenic composition isbuffered from pH 6.5 to 7.5, or to about pH 7.0. In some embodiments,the immunogenic composition further comprises a surfactant. In a subsetof these embodiments, the surfactant comprises polysorbate. In someembodiments, the immunogenic composition further comprises an additionaladjuvant. In a subset of these embodiments, the additional adjuvant iscomprises alum. In some preferred embodiments, the HBsAg is arecombinant HBsAg produced in yeast. In some preferred embodiments, theHBsAg comprises the S antigen. In other preferred embodiments, the HBsAgfurther comprises one or both of the pre-S2 antigen, and the pre-S1antigen. In some embodiments, the HBsAg antigen is purified from plasmaof an HBV-infected subject. In other embodiments, the HBsAg antigen is arecombinant HBsAg produced in mammalian cells in vitro. Furthervariations of these methods are described in the preceding paragraphs.

Furthermore, the present disclosure provides methods for eliciting adurable immune response against hepatitis B virus (HBV) in ahypo-responsive human population according to an abbreviated vaccinationschedule, comprising: administering to the hypo-responsive humanpopulation an immunogenic composition according to an abbreviatedvaccination schedule, wherein the abbreviated vaccination schedulecomprises administering a first, second and third dose of theimmunogenic composition on three separate occasions, wherein theimmunogenic composition comprises a hepatitis B surface antigen (HBsAg)and an immunostimulatory sequence (ISS) of from 8 to 50 nucleotides inlength comprising an unmethylated cytosine-phosphate-guanosine (CpG)motif, and wherein the HBsAg and the ISS are present in the immunogeniccomposition in amounts effective to elicit a durable immune responsecomprising an anti-HBsAg geometric mean concentration of at least 50,75, 100, 150, 200 or 250 mUI/mL the hypo-responsive population 6 monthsor later after the third dose. Also provided are methods for eliciting adurable immune response against hepatitis B virus (HBV) in a humansubject of a hypo-responsive population according to an abbreviatedvaccination schedule, comprising: administering to the human subject animmunogenic composition according to an abbreviated vaccinationschedule, wherein the abbreviated vaccination schedule comprisesadministering a first, second and third dose of the immunogeniccomposition on three separate occasions (e.g., first dose at week 0, asecond dose at week 4, and a third dose at week 24), wherein theimmunogenic composition comprises a hepatitis B surface antigen (HBsAg)and an immunostimulatory sequence (ISS) of from 8 to 50 nucleotides inlength comprising an unmethylated cytosine-phosphate-guanosine (CpG)motif, and wherein the HBsAg and the ISS are present in the immunogeniccomposition in amounts effective to elicit a durable immune responsecomprising an anti-HBsAg concentration of at least 50, 75, 100, 150, 200or 250 mUI/mL in the human subject 6 months or later after the thirddose. In some embodiments, the hypo-responsive population is selectedfrom the group consisting of older adults, males, obese individuals,smokers, type I diabetics, type II diabetics, patients with chronickidney disease, patients with chronic liver disease, patients receivingimmunosuppressive therapy (transplant recipients), patients receivingchemotherapy (cancer patients), HIV-infected individuals, andHCV-infected individuals. In some embodiments, the durable immuneresponse comprises an anti-HBsAg concentration of at least 100 mIU/mL inthe human subject at one month after the third dose. In someembodiments, the hypo-responsive population comprises one or more ofolder adults, males, obese individuals, smokers, diabetics (type Idiabetics and type II diabetics) and patients with chronic kidneydisease. In some embodiments, the human subject has chronic kidneydisease. In some embodiments, the human subject has chronic kidneydisease and diabetes. In some embodiments, the ISS comprises thenucleotide sequence 5′-TCG-3′. In some embodiments, the ISS comprisesthe nucleotide sequence ‘5-CGTTCG-3’ or ‘5-AACGTTCG-3’. In someembodiments, the ISS comprises the nucleotide sequence of SEQ ID NO:1.In other embodiments, the ISS comprises the nucleotide sequence of SEQID NO:2. In some preferred embodiments, the ISS comprises a phosphatebackbone modification. In some preferred embodiments, the ISS comprisesa phosphorothioate backbone modification. In some embodiments, the ISSis single-stranded. In some embodiments, the ISS is double-stranded. Insome preferred embodiments, the ISS is 1018 ISS. In some embodiments,the immunogenic composition comprises 20 μg or less (e.g., 0.2-20 μg) ofthe HBsAg. In some embodiments, the immunogenic composition comprises3000 μg or less (e.g., 30-3000 μg) of the ISS. In some preferredembodiments, the immunogenic composition comprises about 20 μg of theHBsAg and about 3000 μg of the ISS. In some embodiments, the immunogeniccomposition further comprises a buffer. In some embodiments, the buffercomprises sodium phosphate and sodium chloride. In some preferredembodiments, the immunogenic composition is buffered from pH 6.5 to 7.5,or to about pH 7.0. In some embodiments, the immunogenic compositionfurther comprises a surfactant. In a subset of these embodiments, thesurfactant comprises polysorbate. In some embodiments, the immunogeniccomposition further comprises an additional adjuvant. In a subset ofthese embodiments, the additional adjuvant is comprises alum. In somepreferred embodiments, the HBsAg is a recombinant HBsAg produced inyeast. In some preferred embodiments, the HBsAg comprises the S antigen.In other preferred embodiments, the HBsAg further comprises one or bothof the pre-S2 antigen, and the pre-S1 antigen. In some embodiments, theHBsAg antigen is purified from plasma of an HBV-infected subject. Inother embodiments, the HBsAg antigen is a recombinant HBsAg produced inmammalian cells in vitro. Further variations of these methods aredescribed in the preceding paragraphs.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts the level of seroprotection provided by immunization ofdiabetic subjects with HEPLISAV® or ENGERIX-B® respectively.

FIG. 2 depicts the peak level of seroprotection provided by immunizationof various hypo-responsive populations with two doses of HEPLISAV® orthree doses of ENGERIX-B® respectively.

FIG. 3 depicts the level of seroprotection provided by immunization ofsubjects with chronic kidney disease with HEPLISAV® or ENGERIX-B®respectively.

FIG. 4 depicts the level of seroprotection provided by immunization ofsubjects with diabetes and chronic kidney disease with HEPLISAV® orENGERIX-B® respectively.

GENERAL TECHNIQUES

The practice of the present disclosure will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989);Oligonucleotide Synthesis (Gait, ed., 1984); Animal Cell Culture(Freshney, ed., 1987); Handbook of Experimental Immunology (Weir &Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (Miller &Calos, eds., 1987); Current Protocols in Molecular Biology (Ausubel etal., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al.,eds., 1994); Current Protocols in Immunology (Coligan et al., eds.,1991); The Immunoassay Handbook (Wild ed., Stockton Press NY, 1994);Bioconjugate Techniques (Hermanson, ed., Academic Press, 1996); andMethods of Immunological Analysis (Masseyeff, Albert, and Staines, eds.,Weinheim: VCH Verlags gesellschaft mbH, 1993).

DEFINITIONS

As used herein, the singular form “a”, “an”, and “the” includes pluralreferences unless indicated otherwise. For example, “an” excipientincludes one or more excipients.

The phrase “comprising” as used herein is open-ended, indicating thatsuch embodiments may include additional elements. In contrast, thephrase “consisting of” is closed, indicating that such embodiments donot include additional elements (except for trace impurities). Thephrase “consisting essentially of” is partially closed, indicating thatsuch embodiments may further comprise elements that do not materiallychange the basic characteristics of such embodiments.

As used interchangeably herein, the terms “polynucleotide” and“oligonucleotide” include single-stranded DNA (ssDNA), double-strandedDNA (dsDNA), single-stranded RNA (ssRNA) and double-stranded RNA(dsRNA), modified oligonucleotides and oligonucleosides or combinationsthereof. The oligonucleotide can be linearly or circularly configured,or the oligonucleotide can contain both linear and circular segments.Oligonucleotides are polymers of nucleosides joined, generally, throughphosphodiester linkages, although alternate linkages, such asphosphorothioate esters may also be used in oligonucleotides. Anucleoside consists of a purine (adenine (A) or guanine (G) orderivative thereof) or pyrimidine (thymine (T), cytosine (C) or uracil(U), or derivative thereof) base bonded to a sugar. The four nucleosideunits (or bases) in DNA are called deoxyadenosine, deoxyguanosine,thymidine, and deoxycytidine. A nucleotide is a phosphate ester of anucleoside.

The term “immunostimulatory sequence” or “ISS” as used herein refers toa CpG-containing oligonucleotide in which the C is unmethylated, andwhich contributes to a measurable immune response as measured in vitro,in vivo and/or ex vivo. Examples of measurable immune responses include,but are not limited to, antigen-specific antibody production, secretionof cytokines, activation or expansion of lymphocyte populations such asNK cells, CD4+ T lymphocytes, CD8+ T lymphocytes, B lymphocytes, and thelike. Preferably, the ISS preferentially activates a Th1-type response.

An “effective amount” or a “sufficient amount” of a substance is thatamount sufficient to effect beneficial or desired results, includingclinical results, and, as such, an “effective amount” depends upon thecontext in which it is being applied. In the context of administering animmunogenic composition, an effective amount contains sufficient ISS andHBsAg to elicit an immune response (preferably a seroprotective level ofantibody to HBsAg or anti-HBsAg). An effective amount can beadministered in one or more doses.

The term “about” as used herein in reference to a value, encompassesfrom 90% to 110% of that value (e.g., about 20 μg HBsAg refers to 1.8 μgto 22 μg HBsAg).

The term “dose” as used herein in reference to an immunogeniccomposition refers to a measured portion of the immunogenic compositiontaken by (administered to or received by) a subject at any one time.

As used herein the term “immunization” refers to a process thatincreases an organisms' reaction to antigen and therefore improves itsability to resist or overcome infection.

The term “vaccination” as used herein refers to the introduction ofvaccine into a body of an organism.

The phrase “abbreviated vaccination schedule” as used herein refers to avaccination time table that is shorter than a standard vaccinationschedule (e.g., fewer total doses taken over the same or a shorter timeperiod). For instance, an abbreviated vaccination schedule is a two doseschedule involving administration of an immunogenic composition at month0 and month 1, as compared to a standard three dose vaccination scheduleinvolving administration of an immunogenic composition at month 0, month1 and month 6. In another embodiment, an abbreviated vaccinationschedule is a three dose schedule involving administration of animmunogenic composition at month 0, month 1 and month 6, as compared toa standard four dose vaccination schedule involving administration of animmunogenic composition at month 0, month 1, month 2 and month 6.

“Adjuvant” refers to a substance which, when added to a compositioncomprising an antigen, nonspecifically enhances or potentiates an immuneresponse to the antigen in the recipient upon exposure.

The condition of “hyperglycemia” (high blood sugar) is a condition inwhich the blood glucose level is too high. Typically, hyperglycemiaoccurs when the fasting blood glucose level rises above 180 mg/dl.Symptoms of hyperglycemia include frequent urination, excessive thirstand, over a longer time span, weight loss.

On the other hand, “hypoglycemia” (low blood sugar) is a condition inwhich the blood glucose level is too low. Typically, hypoglycemia occurswhen the blood glucose level falls below 70 mg/dl. Symptoms ofhypoglycemia include moodiness, numbness of the extremities (especiallyin the hands and arms), confusion, shakiness or dizziness.

The term “impaired glucose tolerance” is used to describe a person who,when given a glucose tolerance test, has a blood glucose level thatfalls between normal and hyperglycemic. Such a person is at a higherrisk of developing diabetes, although they are not considered to havediabetes.

The term “glucose non-responsive” as used herein describes both thecomplete inability of cells, or islets to respond to treatment with oradministration of glucose, as well as decreased responsiveness toglucose (e.g., by cells that do not produce sufficient levels of insulinin response to glucose or that require significantly higher levels ofglucose to respond at normal levels).

DETAILED DESCRIPTION

The present invention relates to immunization of hypo-responsive groupsof individuals. In particular, the present invention provides methodsand compositions for eliciting a potent immune response to hepatitis Bvirus in subjects having a glucose metabolism disorder.

HEPLISAV® (recombinant HBsAg+1018 ISS vaccine developed by DynavaxTechnologies Corporation) has been shown to produce a more rapid, highertiter, and sustained seroprotective antibody response in healthy adultsas compared to a currently licensed hepatitis B vaccine (Barry andCooper, Expert Opin Biol Ther, 7:1731-1737, 2007; Halperin et al.,Vaccine, 21:2461-2467, 2003; Halperin et al., Vaccine, 24:20-26, 2006;Madaan, Drugs of the Future, 34:531-535, 2009; and Sung and Chan, CurrOpin Molec Ther, 8:150-155, 2006). Other CpG-containingoligodeoxynucleotides (ODN) have been shown to improve theimmunogenicity of hepatitis B virus (HBV) vaccines in preclinical andclinical studies (Cooper et al., CID, 46:1310-1314, 2008; and Payette etal., Intervirology, 49:144-151, 2006).

Although ODN-containing adjuvants appear to improve the immune responsein some populations of hypo-responsive subjects (e.g., older healthyadults, and patients with end stage renal disease), during developmentof the present disclosure, HEPLISAV was found to remarkably improve theimmune response to HBsAg in diabetic subjects, as well as otherhypo-responsive populations (older adults, males, obese, smokers, andpatients with chronic kidney disease). In particular, the presentdisclosure is based on the surprising finding that HEPLISAV induces aseroprotective anti-HBsAg response (defined as an anti-HBsAg level of≧10 mIU/mL) in diabetic subjects after only two doses. HEPLISAV alsoinduces a seroprotective anti-HBsAg response in adults, males, obese,smokers, and patients with chronic kidney disease after only two doses.Specifically, HEPLISAV given as two doses over one month demonstrated asuperior seroprotection rate as compared to a licensed HBV vaccine givenas three doses over six months. Additionally, HEPLISAV induces aseroprotective anti-HBsAg response in patients with chronic kidneydisease (CKD) including CKD patients with type II diabetes after twodoses, and a high titer anti-HBsAg response (100 mIU/ml or greater)after three doses. This observation is in stark contrast to publishedrecommendations for administration of four double doses containing twicethe concentration of HBsAg to hypo-responsive populations.

Hypo-Responsive Subjects

Microbial infections cause an increase in morbidity and mortality inseveral patient populations. For instance, influenza virus infection ismore likely to cause serious disease in the elderly, patients havingpre-existing cardiovascular, renal, diabetic or pulmonary disease, andimmunocompromised individuals (Dorrell et al., International Journal ofSTD & AIDS, 8:776-770, 1997). Individuals with diabetes mellitus have ahigher incidence of infection than non-diabetic individuals. Theincrease in susceptibility to infection by diabetics is in large part aconsequence of defects in their immune response (Geerlings andHoepelman, FEMS Immunol Med Microbiol, 26:259-265, 1999). Importantly,diabetics in assisted care facilities are at increased risk of HBVinfection (Tohme et al., Vaccine, 29:9316-9320, 2011), which highlightsthe need for an effective vaccine for these individuals.

Diabetic subjects have been reported to mount a suboptimal immuneresponse following hepatitis B vaccination (Pozzilli et al.,Diabetologia, 30:817-819, 1987; and Alavian and Tabatabaei, Vaccine,28:3773-3777, 2010). For this reason, supplementary hepatitis Bvaccinations are recommended for diabetic patients (Douvin et al.,Diabetes Care, 20:148-151, 1997; and Wismans et al., J Med Virol,35:216-222, 1991). Similarly, double doses of an HBV vaccine and/or afurther booster are indicated for patients with renal disease (Beran,Expert Opin Biol Ther, 8:235-247, 2008; and Alavian and Tabatabaei,supra, 2010).

The present disclosure provides methods and compositions for inducing aseroprotective immune response to hepatitis B virus surface antigen inhyporesponsive subjects. In some embodiments, the hypo-responsivesubject is an individual with a glucose metabolism disorder. In someembodiments, the glucose metabolism disorder is type I diabetes, type IIdiabetes, or pre-diabetes. In some embodiments, the hypo-responsivesubject is a member of one or more of the groups consisting of olderadults (40 years of age or older), males, obese individuals (body massindex of 30 kg/m² or greater), smokers, patients with chronic kidneydisease (glomerular filtration rate of 45 mL/min/1.73 m² or less) anddiabetics. In some embodiments, the hypo-responsive subject is HIV+and/or HCV+. In some embodiments, the hypo-responsive individual haschronic liver disease. In some embodiments, the hypo-responsive subjectis receiving immunosuppressive therapy or chemotherapy at the onset ofthe administration of the immunogenic composition. In some embodiments,the hypo-responsive subject is a member of multiple hypo-responsivegroups.

As used herein in connection with groups of individuals, the term“hypo-responsive” refers to groups of people that are known to mountsubstantially inferior immune responses to a subunit vaccine incomparison to a normal group of study subjects. An exemplary normalgroup of study subjects are healthy young adults (e.g., under the age of40). As used herein in reference to a group of individuals, the term“population” refers to at least 10, 25, 50, 100, 250, 500, 1,000 or moreindividuals who share a given characteristic (e.g., smokers). As usedherein, the term “population” refers to a plurality of individuals, butdoes not require that the individuals live in the same locale.Additionally in reference to the methods of the present disclosure, thephrase “administering to a population” does not require that thepopulation receive the immunogenic composition at the same locale or atthe same time. That is the individuals of the defined population simplyreceive the defined immunogenic composition according to the definedimmunization schedule.

Diabetes mellitus is a heterogeneous group of metabolic diseases thatlead to chronic elevation of glucose in the blood (hyperglycemia).Diabetes is characterized by pancreatic islet destruction or dysfunctionleading to loss of glucose regulation. The two major types of diabetesmellitus are type I diabetes, also known as insulin-dependent diabetes(IDDM) or juvenile-onset diabetes, and type II diabetes, also known asnon-insulin dependent (NIDDM) or adult-onset diabetes. Subjects with aplasma glucose level of greater than or equal to 11.1 mmol/L (200 mg/dL)two hours after a 75 g oral glucose load as in a glucose tolerance testare considered to be diabetic.

Type I diabetes results from an autoimmune-mediated destruction ofpancreatic beta-cells. This results in a loss of insulin production andhyperglycemia. Type I diabetics require insulin replacement therapy toregulate their blood glucose levels.

Type II diabetes, in contrast, is characterized by hyperglycemia in thepresence of higher-than-normal levels of plasma insulin(hyperinsulinemia). Type II diabetes represents over 90% of all cases,and occurs most often in overweight adults over 40 years of age.Progression of type II diabetes is associated with an increase in bloodglucose, coupled with a relative decrease in the rate of glucose-inducedinsulin secretion. In type II diabetes, physiological processes thatcontrol carbohydrate metabolism are thought to have decreasedsensitivity to insulin. Thus, treatment of type II diabetes frequentlydoes not require administration of insulin, but may instead be based ondiet and lifestyle changes, augmented by therapy with oral hypoglycemicagents.

Pre-diabetes is a condition in which blood glucose levels are higherthan normal, yet below that for a diagnosis of diabetes. This conditionis sometimes called impaired fasting glucose (IFG) or impaired glucosetolerance (IGT), depending on the test used to diagnose it. People withpre-diabetes are at increased risk of developing type II diabetes,formerly called adult-onset diabetes or noninsulin-dependent diabetes.Subjects with fasting glucose levels from 100 to 125 mg/dL (5.6 to 6.9mmol/L) are considered to have impaired fasting glucose, while subjectswith plasma glucose at or above 140 mg/dL (7.8 mmol/L), but not over 200mg/dL (11.1 mmol/L), two hours after a 75 g oral glucose load areconsidered to have impaired glucose tolerance.

Injectable insulin replacement therapy utilizes one or more ofrapid-acting, short-acting, intermediate-acting and long-acting insulinformulations (WebMD at “diabetes.webmd.com/diabetes-types-insulin”).Rapid-acting insulin formulations include but are not limited toHumalog, Lispro, Novolog, Aspart, Apidra and Glulisine. Short-actinginsulin formulations include but are not limited to Humulin, Novolin andVelosulin. Intermediate-acting insulin formulations include but are notlimited to NPH(N) and Lente (L). Long-acting insulin formulationsinclude but are not limited to Ultralente (U), Lantus, Levemir andDetemir.

Oral hypoglycemic agents for treatment of type II diabetes include butare not limited to biguanides, sulfonylureas, meglitinides,thiazolidinediones, and alpha-glucosidase inhibitors (U.S. Dept. HealthHuman Services at “www.ahrq.gov”). Metformin is a biquanide marketed asGlucophage. Sulfonylureas include but are not limited to tobutamide,acetohemamide, tolazamide, chlorpropamide, glimepiride, glipizide,glyburide and gliclazide. Meglitinides include but are not limited torepaglinide and nateglinide. Thiazolidinediones include but are notlimited to pioglitazone and rosiglitazone. Alpha-glucosidase inhibitorsinclude but are not limited to acarbose and miglitol.

Immunostimulatory Sequences (ISS)

Bacterial DNA possesses immunostimulatory properties absent invertebrate DNA. These properties are related to the higher frequency ofunmethylated cytosine-phosphate-guanosine (CpG) motifs and to theabsence of cytosine methylation in bacterial DNA (Bird, Nature,321:209-213, 1986: and Pisetsky, Immunity, 5:303-310, 1996). The effectsof bacterial DNA can be mimicked using synthetic oligonucleotides.Optimal immunostimulatory sequences (ISS) often contain palindromichexamers following the general formula of:5′-purine-purine-CG-pyrimidine-pyrimidine-3′, (Tokunaga et al.,Microbiol Immunol, 36:55-66, 1992; and Yamamoto et al., J Immunol,148:4072-4076, 1992). ISS activity is also noted with certainnon-palindromic CpG-enriched phosphorothioate oligonucleotides. ISSactivity may be affected by changes in the nucleotide sequence. Forinstance, ISS activity is abolished by cytosine methylation of the Cwithin the CpG dinucleotide.

Bacterial DNA and synthetic oligonucleotides containing ISS havemultiple effects on the immune system. These include induction of B-cellproliferation and immunoglobulin production, secretion of interferon(IFN)-α, IFN-β, interleukin (IL)-12, and IL-18 by macrophages and IFN-γsecretion from natural killer cells (Krieg et al., Nature, 374:546-549,1995; Klinman et al., Proc Natl Acad Sci USA, 93:2879-2883, 1996; andMessina et al., J Immunol, 147:1759-1764, 1991; Sato et al., Science,273:352-354, 1996; Yamamoto et al., Japn J Cancer Res, 79:866-873, 1988;Halpern et al., Cell Immunol, 167:72-78, 1996; Roman et al., Nat Med,3:849-854, 1997; and Cowdery et al., J Immunol, 156:4570-4575, 1996).Therefore, ISS appears to stimulate the innate immune system to produceIFN-γ and inducers of IFN-γ (IFN-α, IFN-β, IL-12 and IL-18) and foster acytokine milieu that greatly facilitates the induction of T cells thatprovide help for antibody production, especially those of the T-helper 1(Th1) phenotype.

Researchers at Dynavax (Berkeley, Calif.) have identified a 22-merphosphorothioate 2′-deoxyribonucleotide, 1018 ISS, that contains aspecific sequence that can substantially enhance the immune response toco-administered antigens. 1018 ISS was chosen after screening a broadpanel of oligonucleotides for immunostimulatory activity in vitro and invivo. 1018 ISS (5′-TGACTGTGAA CGTTCGAGAT GA-3′, set forth as SEQ IDNO:1) is active in mice, rabbits, dogs, baboons, cynomolgus monkeys andin vitro in human cells. Co-administration of 1018 ISS with proteinantigens profoundly influences the magnitude and quality of the immuneresponse to the antigens, including an increase in the overall antibodyresponse to antigens. Consistent with this Th1-type response, 1018 ISSalso significantly increases cytotoxic T lymphocyte (CTL) responses toprotein antigens (Cho et al., Nat Biotechnol, 18:509-514, 2000; andTighe et al., Eur J Immunol, 30:1939-1947, 2000).

The methods and compositions of the present disclosure comprise an ISScomprising an unmethylated cytosine-phosphate-guanosine (CpG) motif. Insome preferred embodiments, the ISS comprises TCG in which the C isunmethylated, and which is from 8 to 100 nucleotides, preferably 8 to 50nucleotides, or preferably 8 to 25 nucleotides in length. In someembodiments, the ISS comprises TCG in which the C is unmethylated, andwhich is from 8 to 200 nucleotides (bases or base pairs) in length. Insome embodiments, the ISS is at least 8, 10, 15, or 20 nucleotides inlength and is less than 50, 75, 100, 125, 150, 175 or 200 nucleotides inlength. In some embodiments, the ISS is a 1018 ISS or a 1018 ISS-likeoligonucleotide. The 1018 ISS consists of 22 nucleotides. The 1018-likeISS comprises 5′-AACGTTCG-3′. In some embodiments, the 1018-like ISS isat least 10, 15, or 20 nucleotides in length. In some preferredembodiments, the 1018 ISS-like oligonucleotide is less than 100nucleotides in length, preferably less than 50, 40 or 30 nucleotides inlength. For the sake of brevity, the CpG-containing ISS, theTCG-containing ISS, the 1018 ISS and the 1018 ISS-like oligonucleotidesof the present disclosure are referred to below simply as an “ISS of thepresent disclosure” or “ISS.” In some embodiments, the ISS issingle-stranded, while in other embodiments, it is double-stranded. Insome preferred embodiments, the ISS comprises a phosphate backbonemodification. In some preferred embodiments, the ISS comprises aphosphorothioate backbone modification. In some embodiments, the ISScomprises 5′-TCGTCGTTTT GTCGTTTTGT-CGTT-3′ (SEQ ID NO:2).

Specifically, an ISS of the present disclosure may containmodifications. Modifications include any known in the art, but are notlimited to, modifications of the 3′OH or 5′OH group, modifications ofthe nucleotide base, modifications of the sugar component, andmodifications of the phosphate group. Modified bases may be included inthe palindromic sequence of the ISS as long as the modified base(s)maintains the same specificity for its natural complement throughWatson-Crick base pairing (e.g., the palindromic portion is stillself-complementary). The ISS may be linear, circular or include circularportions and/or a hairpin loop. The ISS may be single stranded or doublestranded. The ISS may be DNA or RNA.

The ISS may contain naturally-occurring or modified, non-naturallyoccurring bases, and may contain modified sugar, phosphate, and/ortermini. For example, in addition to phosphodiester linkages, phosphatemodifications include, but are not limited to, methyl phosphonate,phosphorothioate, phosphoramidate (bridging or non-bridging),phosphotriester and phosphorodithioate and may be used in anycombination. Other non-phosphate linkages may also be used. In someembodiments, polynucleotides comprise only phosphorothioate backbones.In some embodiments, polynucleotides comprise only phosphodiesterbackbones. In some embodiments, an ISS may comprise a combination ofphosphate linkages in the phosphate backbone such as a combination ofphosphodiester and phosphorothioate linkages.

An ISS of the present disclosure can be synthesized using techniques andnucleic acid synthesis equipment, which are well known in the artincluding, but not limited to, enzymatic methods, chemical methods, andthe degradation of larger oligonucleotide sequences. Naturally-occurringDNA or RNA, containing phosphodiester linkages, is generally synthesizedby sequentially coupling the appropriate nucleoside phosphoramidite tothe 5′-hydroxy group of the growing oligonucleotide attached to a solidsupport at the 3′-end, followed by oxidation of the intermediatephosphite triester to a phosphate triester. Once the desiredpolynucleotide sequence has been synthesized, the polynucleotide isremoved from the support, the phosphate triester groups are deprotectedto phosphate diesters and the nucleoside bases are deprotected usingaqueous ammonia or other bases (see, e.g., Beaucage,“Oligodeoxyribonucleotide Synthesis” in Protocols for Oligonucleotidesand Analogs, Synthesis and Properties (Agrawal, ed.) Humana Press,Totowa, N.J., 1993).

The ISS can also contain phosphate-modified polynucleotides, some ofwhich are known to stabilize the polynucleotide. Accordingly, someembodiments include stabilized immunomodulatory polynucleotides.Synthesis of polynucleotides containing modified phosphate linkages ornon-phosphate linkages is also known in the art (see, e.g., Matteucci“Oligonucleotide Analogs: an Overview” in Oligonucleotides asTherapeutic Agents, (Chadwick and Cardew, ed. John Wiley and Sons, NewYork, N.Y., 1997). The phosphorous derivative (or modified phosphategroup), which can be attached to the sugar or sugar analog moiety in thepolynucleotides of the present disclosure can be a monophosphate,diphosphate, triphosphate, alkylphosphonate, phosphorothioate,phosphorodithioate, phosphoramidate or the like. The preparation of theabove-noted phosphate analogs, and their incorporation into nucleotides,modified nucleotides and oligonucleotides is known in the art, andtherefore not described here in detail (Peyrottes et al., Nucleic AcidsRes. 24:1841-1848, 1996; Chaturvedi et al., Nucleic Acids Res.24:2318-2323, 1996; and Schultz et al., Nucleic Acids Res. 24:2966-2973,1996). For example, synthesis of phosphorothioate oligonucleotides issimilar to that described above for naturally occurring oligonucleotidesexcept that the oxidation step is replaced by a sulfurization step (Zon“Oligonucleoside Phosphorothioates” in Protocols for Oligonucleotidesand Analogs, Synthesis and Properties (Agrawal, ed.) Humana Press, pp.165-190, 1993). Similarly the synthesis of other phosphate analogs, suchas phosphotriester (Miller et al., JAGS 93:6657-6665, 1971),non-bridging phosphoramidates (Jager et al., Biochem. 27:7247-7246,1988), N3′ to P5′ phosphoramidates (Nelson et al., JOC 62:7278-7287,1997) and phosphorodithioates (U.S. Pat. No. 5,453,496) has also beendescribed. Other non-phosphorous based modified oligonucleotides canalso be used (Stirchak et al., Nucleic Acids Res. 17:6129-6141, 1989).Polynucleotides with phosphorothioate backbones can be more immunogenicthan those with phosphodiester backbones and appear to be more resistantto degradation after injection into the host (Braun et al., J. Immunol.141:2084-2089, 1988; and Latimer et al., Mol. Immunol. 32:1057-1064,1995).

Hepatitis B Surface Antigen (HBsAg)

Methods for preparing HBsAg are well documented (see, Valenzuela et al.,Nature 298:347-350, 1982; U.S. Pat. Nos. 4,710,463, 6,268,122,6,270,955, and 6,297,355 to Murray; U.S. Pat. Nos. 4,769,238, 6,475,489,and 6,544,757 to Rutter et al.). As used herein, the expression“hepatitis B surface antigen” or “HBsAg” includes any HBsAg antigen orfragment thereof displaying the antigenicity of the HBV surface antigen.In addition to the 226 amino acid sequence of the HBsAg S antigen(Tiollais et al., Nature, 317:489, 1985), HBsAg may, if desired, containall or part of a pre-S sequence. HBsAg as used herein may also refer tomutants, for example the “escape mutant” wherein HBsAg comprises asubstitution of glycine to arginine at position 145. In preferredembodiments, the HBsAg is in particle form. In preferred embodiments,the HBsAg is a particle produced recombinantly in yeast. In otherembodiments, the HBsAg is produced recombinantly in mammalian cells. Inother embodiments, the HBsAg is purified from the plasma of an infectedsubject.

Four serotypes or subtypes of the hepatitis B surface antigen (HBsAg)have been defined by common determinant (a) and two mutually exclusivedeterminant pairs (d/y and w/r). These subtypes are adw, ayw, adr andayr (Magnius and Norder, Intervirology, 38:24-34, 1995). The immunogeniccompositions of the present disclosure are suitable for immunizing ahypo-responsive subject against infection caused by all subtypes of HBV.

Immunogenic Compositions and Administration Thereof

The immunogenic compositions for use with the methods disclosed herein,comprise 1018 ISS or a 1018 ISS-like oligonucleotide and a hepatitis Bvirus surface antigen. The immunogenic compositions may further comprisean additional adjuvant and/or a pharmaceutically acceptable excipient.Pharmaceutically acceptable excipients, including buffers, are wellknown in the art (see, e.g., Remington: The Science and Practice ofPharmacy, 20th edition, Mack Publishing, 2000).

Upon administration, compositions comprising an antigen, 1018 ISS or a1018 ISS-like oligonucleotide, and optionally an additional adjuvantlead to a potentiation of an immune response to the antigen. Adjuvantsare known in the art and include, but are not limited to, oil-in-wateremulsions, water-in oil emulsions, alum (aluminum salts), liposomes andmicroparticles, including but not limited to, polystyrene, starch,polyphosphazene and polylactide/polyglycosides. Other suitable adjuvantsalso include, but are not limited to, MF59, DETOX™ (Ribi), squalenemixtures (SAF-1), muramyl peptide, saponin derivatives, mycobacteriumcell wall preparations, monophosphoryl lipid A, mycolic acidderivatives, nonionic block copolymer surfactants, Quil A, cholera toxinB subunit, polyphosphazene and derivatives, and immunostimulatingcomplexes (Takahashi et al., Nature 344:873-875, 1990), as well asothers described herein. For veterinary use and for production ofantibodies in non-human animals, mitogenic components of Freund'sadjuvant (both complete and incomplete) can be used.

As with all immunogenic compositions, the immunologically effectiveamounts and method of administration of the particular formulation canvary based on the individual, what condition is to be treated and otherfactors evident to one skilled in the art. One factor to be consideredincludes the antigenicity of antigen, whether or not the ISS isadministered in a mixture with, non-covalently associated with orcovalently attached to the antigen. Other factors to be considered arethe route of administration, number of doses to be administered, andtime period between doses. A suitable dosage range is one that providesthe desired modulation of immune response (e.g., stimulation of aseroprotective anti-HBsAg response).

In some embodiments, the immunogenic composition comprises from 1 μg to50 μg HBsAg, preferably 4 to 40 μg HBsAg. In some preferred embodiments,the immunogenic composition comprises from 5 μg to 25 μg HBsAg (e.g., 5,10, 15, 20 or 25 μg HBsAg), or more preferably from 10 μg to 20 μg HBsAg(e.g., 10, 15, or 20 μg HBsAg). In an exemplary embodiment, theimmunogenic composition comprises 20 μg HBsAg. In some embodiments, theimmunogenic composition comprises from 100 μg to 5000 μg of 1018 ISS or1018 ISS-like oligonucleotide. In some preferred embodiments, theimmunogenic composition comprises from 300 μg to 3000 μg, or morepreferably from 500 μg to 5000 μg ISS (e.g., 500, 1000, 1500, 2000,2500, 3000, 3500, 4000, 4500 or 5000 μg ISS), or more preferably 1000 μgto 3000 μg ISS (e.g., 1000, 1500, 2000, 2500, 3000 μg ISS). In somepreferred embodiments, the immunogenic composition comprises about 20 μgHBsAg and about 3000 μg ISS.

Routes of administration include but are not limited to topical, dermal,transdermal, transmucosal, epidermal, subcutaneous, parenteral,gastrointestinal, and naso-pharyngeal and pulmonary, includingtransbronchial and transalveolar. In a preferred embodiment, theimmunogenic composition is administered by intradermal injection. In apreferred embodiment, the immunogenic composition is administered byintramuscular injection.

In some embodiments, the immunogenic compositions of the presentdisclosure comprise HBsAg and a further antigen. In some preferredembodiments, the further antigen is an inactivated hepatitis A virus. Inadditional embodiments, the further antigen comprises one or more of thegroup consisting of an inactivated hepatitis A virus, a diphtheriatoxoid, a tetanus toxoid, acellular pertussis, Haemophilus influenzaeType B (HiB), an inactivated polio virus, and an influenza virus (FLU)Particular combination vaccines within the scope of the presentdisclosure include: Diphtheria-Tetanus-Pertussis-Hepatitis B (DTP-HB),Diphtheria-Tetanus-Hepatitis B (DT-HB), andDiphtheria-Tetanus-Pertussis-Haemophilus-Hepatitis B (DTP-HiB-HB).Additional combinations include: Influenza-Hepatitis B (FLU-HB), andHepatitis A-Hepatitis B (HA-HB) Other combination vaccines of thepresent disclosure include Human Papilloma Virus (HPV) antigen-HepatitisB (HPV-HB), and Varicella Zoster Virus (VZV)-Hepatitis B (VZV-HB).

In some embodiments, the present disclosure provides kits thatcomprising an immunogenic composition and a set of instructions relatingto the use of the immunogenic composition for the methods describeherein. The kits may comprise an immunogenic composition packagedappropriately. For example, if the immunogenic composition isfreeze-dried power, a vial with a resilient stopper is normally used sothat the powder may be easily resuspended by injecting fluid through theresilient stopper. In some embodiments, the kits comprise a device foradministration (e.g., syringe and needle). The instructions relating tothe use of the immunogenic composition generally include information asto dosage, schedule and route of administration for the intended methodsof use.

EXAMPLES

Abbreviations: GMC (geometric mean concentration); HBcAb or anti-HBc(hepatitis B core antibody); HBcAg (hepatitis B core antigen); antibodyto HBsAg, anti-HBsAg, or anti-HBs (hepatitis B surface antibody); HBsAg(hepatitis B surface antigen); HBV (hepatitis B virus); HEPLISAV(recombinant HBsAg+1018 ISS vaccine of Dynavax); ISS (immunostimulatorysequence); ITT (intent-to-treat population); mITT (modified intent totreat population); mIU/mL (milli international units/milliliter); PP(per-protocol); and SPR (seroprotective immune response, defined as[anti-HBsAg] of ≧10 mIU/mL).

Example 1 Immunogenicity of Two Doses of HEPLISAV Compared to ThreeDoses of an Approved Recombinant HBsAg Vaccine

This example provides a description of a multicenter, phase threeclinical study conducted among healthy adults, which compared theimmunogenicity of two doses of HEPLISAV (HBsAg+1018 ISS vaccine ofDynavax, Berkeley, Calif.) administered over 4 weeks to three doses ofENGERIX-B (HBsAg adsorbed to alum vaccine of GlaxoSmithKline, ResearchTriangle Park, N.C.) administered over 24 weeks. An ad-hoc analysis wasconducted to compare the immunogenicity of these two vaccine regimensamong persons with type II diabetes. Diabetes was assessed by the reviewof recorded subject medical history and prior/concomitant use of oralhypoglycemics and/or insulin. Additionally an ad-hoc analysis wasconducted to compare the immunogenicity of these two vaccine regimensamong further hypo-responsive populations.

Primary Immunogenicity Objective: To compare the proportion of subjectswho exhibit seroprotective immune response (SPR=anti-HBsAg antibodylevel ([anti-HBsAg])≧10 mIU/mL) when measured at Week 12 followingvaccination with HEPLISAV at months 0 and 1 to the proportion ofsubjects who exhibit SPRs when measured at Week 28 following vaccinationwith ENGERIX-B at months 0, 1 and 6.

Secondary Immunogenicity Objective(s): To compare the proportion ofsubjects exhibiting an SPR for HEPLISAV versus ENGERIX-B when measuredat Week 4.

Exploratory Immunogenicity Objective(s): To compare the proportion ofsubjects exhibiting an SPR for HEPLISAV versus ENGERIX-B when measuredat Weeks 8, 12, 24 and 28. To describe the anti-HBsAg serum geometricmean concentrations (GMCs) observed for HEPLISAV and ENGERIX-B whencalculated at Weeks 4, 8, 12, 24 and 28 (durability of response). Tocompare the proportion of subjects who exhibit seroprotective immuneresponse (SPR=[anti-HBsAg]≧10 mIU/mL) when measured at Week 8 followingvaccination with HEPLISAV at Weeks 0 and 4 to the proportion of subjectswho exhibit SPR when measured at Week 28 following vaccination withENGERIX-B at Weeks 0, 4, and 24.

Study Design—Diabetic Subjects.

This study was conducted as a subject- and observer-blinded, randomized,controlled study of adult subjects (ages 11 to 55 years) randomized 3:1to receive injections with either HEPLISAV (3000 μg 1018 ISS+20 μgrHBsAg) or ENGERIX-B (20 μg rHBsAg in alum). Subjects were stratified byage (11 to 39 years of age versus 40 to 55 years of age) prior torandomization.

Study Design—Other Hypo-Responsive Subjects.

This study was conducted as a subject- and observer-blinded, randomized,controlled study of adult subjects (ages 40 to 70 years) randomized 3:1to receive injections with either HEPLISAV (3000 μg 1018 ISS+20 μgrHBsAg) or ENGERIX-B (20 μg rHBsAg in alum). Subjects were stratifiedgender, body mass index (BMI ≧30 kg/m² as obese versus BMI <30 kg/m² asnon-obese), and nicotine use (smokers versus non-smokers) prior torandomization.

Diabetic and other hypo-responsive subjects received a total of threeintramuscular injections (active vaccine or matching placebo), given onstudy Day 0, Week 4 (1 month), and Week 24 (6 month). Subjectsrandomized to ENGERIX-B received three injections of ENGERIX-B (20 μgrHBsAg adsorbed to alum) at Weeks 0, 4 and 24. Subjects randomized toHEPLISAV received an injection of 3000 μg 1018 ISS+20 μg rHBsAg at Weeks0 and 4, and a saline placebo at Week 24. All subjects were asked toreturn approximately 4 weeks after each injection to have blood drawn tomeasure anti-HBsAg levels and to undergo safety evaluations.

Study Population.

Subjects were selected from among HBV seronegative male and femalevolunteers. Inclusion and exclusion criteria met by study participantsincluded but were not limited to the listing provided below. InclusionCriteria: serum negative for HBsAg, anti-HBsAg and anti-HBcAg. ExclusionCriteria: history of HBV infection; prior immunization with any HBVvaccine; clinically debilitating illness, (e.g., fever ≧38° C. within 72hours prior to study injection, bleeding disorders, cancer, autoimmunedisease, immunodeficiency, etc.); history or laboratory evidence ofautoimmune disease; high risk for recent exposure to HBV or HIV; recentreceipt of blood products or likely to require infusion of bloodproducts; previously received DNA plasmids or oligonucleotides byinjection; and recent use of systemic corticosteroids, otherimmunomodulators or other immunosuppressive medications (with theexception of inhaled steroids); and history of sensitivity to anycomponent of the study vaccines.

Subjects included in the diabetes cohort were selected based on amedical history including preferred terms with DIABET, and excludingGESTATIONAL DIABETES. Additional subjects in the diabetes cohort wereselected based on the use of concomitant medication (ATC code A10) fordiabetes, in the absence of a documented medical history of diabetes.

Treatments Administered.

HEPLISAV (3000 μg 1018 ISS+20 μg rHBsAg) was manufactured by RentschlerBiotechnologie GmbH, Laupheim, Germany for Dynavax. TechnologiesCorporation, Berkeley, Calif.) The rHBsAg of this formulation wasderived from yeast cells transformed with an expression vectorcontaining HBsAg (S) sequence, subtype adw. 1018 ISS is asingle-stranded, 22-base phosphorothioate 2′-deoxyribo-oligonucleotideprepared by standard solid-phase chemistry techniques (5′-TGACTGTGAACGTTCGAGAT GA-3′, set forth as SEQ ID NO:1). 1018 ISS has a molecularmass of approximately 7150 Daltons. HEPLISAV also contains the followingexcipients: 8 mM sodium phosphate, 154 mM sodium chloride, and 0.01% w/wpolysorbate 80, pH 7.0 buffer. The HEPLISAV drug product is formulatedas 6000 mcg/mL 1018 ISS and 40 mcg/mL HBsAg in a 2-mL vial containing0.7 mL of solution (28 mcg of protein and 4200 mcg of 1018 ISS per vial)of which a 0.5 mL dose (20 mcg of protein and 3000 mcg of 1018 ISS) isadministered. HEPLISAV is stored at 2 to 8° C. before use.

Immunogenicity Analyses.

Two patient populations were considered for the immunogenicity analysis:the per-protocol (PP) population and the intent-to-treat (ITT)population. The immunogenicity analysis using the PP population wasconsidered primary. PP Population-Immunogenicity: The PP populationincluded subjects who met the eligibility criteria, did not violate theprotocol in a substantial manner, received all protocol-specified studyvaccinations, had their primary serology and all vaccinations within thespecified day ranges, and had serology at their primary endpoint (week12 for HEPLISAV and week 28 for ENGERIX-B). ITTPopulation-Immunogenicity: The ITT population included subjects who hadat least one vaccination and one post baseline anti-HBsAg level.Anti-HBsAg was measured by using the hepatitis B enhancedchemiluminescence immunoassay (Hep B ECi, Ortho Clinical Diagnostics,Rochester, N.Y.).

All statistical tests comparing demographic, patient characteristic andsafety data were two-sided and conducted at the 5% significance level.All immunogenicity analyses utilized one-sided tests at the 2.5% levelof significance. All data analyses were performed using StatisticalAnalysis Systems (SAS) for Windows 95/NT (version 8.2 or later, SASInstitute, Cary, N.C.). The 95% confidence interval (CI) of SPR wascalculated using the Clopper Pearson method. The 95% confidence interval(CI) of the difference (HEPLISAV−ENGRIX-B) was calculated using Newcombescore method with continuity correction. For the immunogenicityobjectives, if the lower bound of 95% CI was greater than −10% thenHEPLISAV was scored as non-inferior. Additionally, if HEPLISAV wasscored as non-inferior, and the lower bound of the 95% CI was greaterthan 0%, then HEPLISAV was also scored as superior.

Seroprotective Immune Response (SPR) Rate.

For the purpose of this analysis, a seroprotective immune response wasdefined as an anti-HBsAg concentration of ≧10 mIU/mL.

Geometric Mean Concentrations (GMCs).

Anti-HBsAg GMC was measured four weeks after each active injection forboth groups. All anti-HBsAg concentrations that were reported as <5.0mIU/mL were considered as 2.5 mIU/mL in the computation for GMC. Log(base 10)-transformed anti-HBsAg concentrations were used to summarizethe GMCs for the two treatment groups.

Results—Diabetic Subjects.

Of the 2101 non-diabetic and diabetic subjects in the overall perprotocol study population (1566 HEPLISAV, and 535 ENGERIX-B), the SPRwas 95% at Week 12 in the HEPLISAV group and 81% at Week 28 in theENGERIX-B group (p<0.001), indicating non-inferiority/superiority ofHEPLISAV. Among the 62 diabetics in the per protocol population, 45 werein the HEPLISAV group (mean age of 44.4 years) and 17 in the ENGERIX-Bgroup (mean age of 45.5 years). Of these subjects, 38 (84%) in theHEPLISAV group achieved SPR compared to 0 (0%) in the ENGERIX-B group atWeek 12 (p<0.0001), and 42 (93%) versus 6 (35%) respectively at Week 28(p<0.0001).

TABLE 1-1 Seroprotection (SPR) Rates of HBsAg Vaccinated StudyPopulations HEPLISA HEPLISA Week V V ENGERIX-B ENGERIX-B Population #Subjects SPR Rate # Subjects SPR Rate P-Value Week 12 PP non- 1,52195.4% 518 23.2% <0.0001 diabetic PP 45 84.4% 17 0.0% <0.0001 diabeticITT 54 85.2% 18 0.0% <0.0001 diabetic Week 28 PP non- 1,520 98.1% 51882.6% <0.0001 diabetic PP 45 93.3% 17 35.3% <0.0001 diabetic ITT 5492.6% 18 33.3% <0.0001 diabetic

TABLE 1-2 Seroprotection (SPR) Rates in Vaccinated Per Protocol DiabeticSubjects HEPLISA HEPLISA Week/ V V ENGERIX-B ENGERIX-B P- Population #Subjects SPR Rate # Subjects SPR Rate Value* Week 4 PP 45 24.4% 17   0%ND diabetic Week 8 PP 45 71.1% 17  5.9% ND diabetic Week 12 PP 45 84.4%17   0% <0.0001 diabetic Week 24 PP 45 93.3% 17 11.8% ND diabetic Week28 PP 45 93.3% 17 35.3% <0.0001 diabetic *ND = Not Done.

TABLE 1-3 Anti-HBsAg Geometric Mean Concentrations (GMC) in VaccinatedPer Protocol Diabetic Subjects+ HEPLISA V HEPLISA V ENGERIX-B ENGERIX-BWeek/Population # Subjects GMC # Subjects GMC P-Value* Week 4 PPdiabetic 45  4.8 17 2.5 ND (3.5, 6.6) (2.5, 2.5) Week 8 PP diabetic 4536.7 17 3.2 ND (21.3, 63.1) (2.2, 4.6) Week 12 PP diabetic 45 47.1 172.8 <0.0001 (29.4, 75.3) (2.4, 3.5) Week 24 PP diabetic 45 109.8  17 3.2ND  (68.2, 176.7) (2.2, 4.7) Week 28 PP diabetic 45 96.9 17 16.7   0.0283  (59.4, 158.3)  (3.8, 74.0) +GMC (95% confidence interval). *ND= Not done.

As determined during development of the present disclosure, in a subsetanalysis of adults with diabetes, HEPLISAV given as two doses over onemonth demonstrated superior SPR compared to ENGERIX-B given as threedoses over six months. Thus, use of HEPLISAV to vaccinate diabeticsprovides superior protection against hepatitis B infection and diseaseas compared to a Food and Drug Administration approved recombinant HBVvaccine.

Results—Other Hypo-Responsive Subjects.

Of the 1482 subjects in the overall per protocol study population (1123HEPLISAV and 359 ENGERIX-B), the peak SPR was 95% in the HEPLISAV groupat week 12 and 73% in the ENGERIX-B group at week 32 with an SPRdifference of 22.3% (P<0.0001), indicating non-inferiority andsuperiority of HEPLISAV. Moreover, the peak SPR of HEPLISAV was superiorto ENGERIX-B in all hypo-responsive subgroups (Table 1-4 and FIG. 2).Within the HEPLISAV group, the SPR was similar between males andfemales, obese and non-obese, and smokers and non-smokers. In theENERIX-B group, the males, obese, and smokers were hypo-responsive incomparison to the females, non-obese, and non-smokers.

TABLE 1-4 Peak % SPR in Per Protocol Populations HEPLISA V ENGERIX BSubjects % SPR (N) % SPR (N) Adults ≧40 yrs 95.1 (1123)^(c) 72.8 (359)Males 94.6 (537)^(c) 67.8 (177) Females 95.6 (586)^(c) 77.8 (180)Obese^(a) 94.7 (494)^(c) 65.4 (153) Non-obese^(b) 95.4 (629)^(c) 78.4(204) Smokers 95.6 (229)^(c) 65.3 (75) Non-smokers 95.0 (894)^(c) 74.8(282) ^(a)BMI ≧ 30 kg/m²; ^(b)BMI < 30 kg/m²; and ^(c)P < 0.0001.

As determined during development of the present disclosure, HEPLISAVgiven as 2 doses over 4 weeks was superior and protected a significantlygreater proportion of subjects than ENGERIX-B given as 3 doses over 24weeks in adults overall and in groups known to be hypo-responsive tocurrently licensed hepatitis B vaccine. A hepatitis B vaccine thatprovides a greater level of seroprotection in hypo-responsive groups isan important public health advancement because it provides protectionfrom HBV to a greater proportion of subjects.

Example 2 Immunogenicity of Three Doses of HEPLISAV Compared to FourDouble Doses of an Approved Recombinant HBsAg Vaccine

This example provides a description of a phase three clinical studyconducted among adults with chronic kidney disease (CKD), which comparedthree doses of HEPLISAV (HBsAg+1018 ISS vaccine of Dynavax, Berkeley,Calif.) administered over six months to four double doses of ENGERIX-B(HBsAg adsorbed to alum vaccine of GlaxoSmithKline, Research TrianglePark, N.C.) administered over six months. CKD was defined by a GFR ofless than or equal to 45 mL/min/1.73 m².

Primary Immunogenicity Objective: To compare the proportion of subjectswho exhibit seroprotective immune response (SPR=anti-HBsAg antibodylevel ([anti-HBsAg])≧10 mIU/mL) when measured at Week 28 followingvaccination with HEPLISAV at months 0, 1 and 6 to the proportion ofsubjects who exhibit SPRs when measured at Week 28 following vaccinationwith ENGERIX-B at months 0, 1, 2 and 6.

Other Immunogenicity Objective(s): To compare the proportion of subjectsexhibiting an SPR for HEPLISAV versus ENGERIX-B at multiple time points.To describe the anti-HBsAg serum geometric mean concentrations (GMCs)observed for HEPLISAV and ENGERIX-B calculated at multiple time points(speed and durability of response).

Study Design.

This study was conducted as a subject- and observer-blinded, randomized,controlled study of adult subjects (ages 18 to 75 years) with chronickidney disease (progressive loss of renal function as defined by GFR ≦45mL/min/1.73 m²) randomized to receive injections with either HEPLISAV(3000 μg 1018 ISS+20 μg rHBsAg) or ENGERIX-B (2×20 μg rHBsAg in alum).Randomization was stratified by GFR (≦15, 16-30, and 31-45 mL/min/1.73m²). All subjects received a total of four intramuscular injections(active vaccine or matching placebo), given on study Day 0 (0 month),Week 4 (1 month), Week 8 (2 month) and Week 24 (6 month). Subjectsrandomized to ENGERIX-B received four double-doses of ENGERIX-B (2×20 μgrHBsAg adsorbed to alum) at Weeks 0, 4, 8 and 24. Subjects randomized toHEPLISAV received three doses of HEPLISAV (3000 μg 1018 ISS+20 μgrHBsAg) at Weeks 0, 4 and 24, and a saline placebo at Week 8. Allsubjects were asked to return approximately 4 weeks after each injectionto have blood drawn to measure anti-HBsAg levels and to undergo safetyevaluations.

Study Population.

Subjects were selected from among HBV seronegative male and femalevolunteers. Inclusion and exclusion criteria met by study participantsincluded but were not limited to the listing provided below. InclusionCriteria: adults with chronic kidney disease, and serum negative forHBsAg, anti-HBsAg and anti-HBcAg. Exclusion Criteria: history of HBVinfection; prior immunization with any HBV vaccine; clinicallydebilitating illness, (e.g., fever ≧38° C. within 72 hours prior tostudy injection, bleeding disorders, cancer, autoimmune disease,immunodeficiency, etc.); high risk for recent exposure to HBV, HCV orHIV; recent receipt of blood products or likely to require infusion ofblood products; previously received DNA plasmids or oligonucleotides byinjection; and recent use of systemic corticosteroids, otherimmunomodulators or other immunosuppressive medications (with theexception of inhaled steroids); and history of sensitivity to anycomponent of the study vaccines.

Treatments Administered.

HEPLISAV (3000 μg 1018 ISS+20 μg rHBsAg) was manufactured by RentschlerBiotechnologie GmbH, Laupheim, Germany for Dynavax. TechnologiesCorporation, Berkeley, Calif.) The rHBsAg of this formulation wasderived from yeast cells transformed with an expression vectorcontaining HBsAg (S) sequence, subtype adw. 1018 ISS is asingle-stranded, 22-base phosphorothioate 2′-deoxyribo-oligonucleotideprepared by standard solid-phase chemistry techniques (5′-TGACTGTGAACGTTCGAGAT GA-3′, set forth as SEQ ID NO:1). 1018 ISS has a molecularmass of approximately 7150 Daltons. HEPLISAV also contains the followingexcipients: 8 mM sodium phosphate, 154 mM sodium chloride, and 0.01% w/wpolysorbate 80, pH 7.0 buffer. The HEPLISAV drug product is formulatedas 6000 mcg/mL 1018 ISS and 40 mcg/mL HBsAg in a 2-mL vial containing0.7 mL of solution (28 mcg of protein and 4200 mcg of 1018 ISS per vial)of which a 0.5 mL dose (20 mcg of protein and 3000 mcg of 1018 ISS) isadministered. HEPLISAV is stored at 2 to 8° C. before use.

Immunogenicity Analyses.

Two patient populations were considered for the immunogenicity analysis:the per-protocol (PP) population and the modified intent-to-treat (mITT)population. Anti-HBsAg was measured by using the hepatitis B enhancedchemiluminescence immunoassay (Hep B ECi, Ortho Clinical Diagnostics,Rochester, N.Y.).

All statistical tests comparing demographic, patient characteristic andsafety data were two-sided and conducted at the 5% significance level.All immunogenicity analyses utilized one-sided tests at the 2.5% levelof significance. All data analyses were performed using StatisticalAnalysis Systems (SAS) for Windows 95/NT (version 8.2 or later, SASInstitute, Cary, N.C.). The 95% confidence interval (CI) of SPR wascalculated using the Clopper Pearson method. The 95% confidence interval(CI) of the difference (HEPLISAV−ENGRIX-B) was calculated using Newcombescore method with continuity correction. For the immunogenicityobjectives, if the lower bound of 95% CI was greater than −10% thenHEPLISAV was scored as non-inferior. Additionally, if HEPLISAV wasscored as non-inferior, and the lower bound of the 95% CI was greaterthan 0%, then HEPLISAV was also scored as superior.

Seroprotective Immune Response (SPR) Rate.

For the purpose of this analysis, a seroprotective immune response wasdefined as an anti-HBsAg concentration of ≧10 mIU/mL.

Geometric Mean Concentrations (GMCs).

Anti-HBsAg concentrations were measured at weeks 0, 4, 8, 12, 18, 24,28, 36, 44 and 52 for both groups. All anti-HBsAg concentrations thatwere reported as <5.0 mIU/mL (limit of the assay) were considered as 2.5mIU/mL in the computation for GMC. Log (base 10)-transformed anti-HBsAgconcentrations were used to summarize the GMCs for the two treatmentgroups.

Results.

The modified intent-to-treat (mITT) population included all subjectswith at least one immunization and a post-immunization antibodyassessment, and consisted of 507 subjects with 247 subjects in theHEPLISAV (H) group and 260 subjects in the ENGRIX-B (EB) group. 63% of Hsubjects and 60% of EB subjects were men. The mean age was 61 years forboth groups. The mean body mass index in kg/m² was 34 for H and 32 forEB. 15% of H and 19% of EB subjects had a GFR ≦15 mL/min/1.73 m². 68% ofH and 61% of EB subjects were diabetic. The incidence of post-injectionreactions and adverse events was similar in both groups. The SPR was89.8% in the H group and 81.8% in the EB group, with an SPR differenceof 8.0% (95% CI: 1.6%, 14.2%), indicating non-inferiority andsuperiority of HEPLISAV. The SPR of HEPLISAV was superior to ENGERIX-Bfrom weeks 8 through 28. The difference in the percentage of subjectswith anti-HBs ≧100 mIU/mL between H and EB was 10.6% (95% CI, 2.1%,18.7%) at month 7. The geometric mean concentration (GMC) in H (589mIU/mL; 95% CI, 387, 896) was significantly higher than the GMC in EB(156 mIU/mL; 95% CI, 104, 236) at month 7, with a 3.8 fold higher GMC inH. In diabetic subjects the SPR in the H group was 89.5% versus 76.7% inthe EB group at month 7, with a SPR difference of 12.8% (95% CI, 4.4%,21.2%), indicating non-inferiority and superiority of H. Further dataanalysis was performed to evaluate the results in CKD patients, as wellas in CKD patients with and without type II diabetes, as shown in Tables2-1 through 2-7 (weeks in bold indicate when vaccine or placeboinjections were administered).

TABLE 2-1 Seroprotection Rates in mITT Subjects with Chronic KidneyDisease without Diabetes HEPLISA V ENGERIX-B % Δ Week % SPR Rate % SPRRate H − E 0 0 0 0 4 1.3 7.0 −5.7 8 61.8 26.5 35.3 12 78.9 57.9 21.1 1884.4 71.3 13.1 24 85.5 71.3 14.2 28 90.4 90.2 0.2 36 91.2 88.2 3.1 4487.5 87.0 0.5 52 85.7 87.5 −1.8

TABLE 2-2 Seroprotection Rates in mITT Subjects with Chronic KidneyDisease with Type II Diabetes HEPLISA V ENGERIX-B % Δ Week % SPR Rate %SPR Rate H − E 0 0.6 0.0 0.6 4 7.7 5.7 2.0 8 41.8 16.4 25.5 12 58.6 46.412.2 18 68.6 52.3 16.3 24 75.5 56.6 18.9 28 89.5 76.7 12.8 36 82.3 76.45.9 44 78.4 73.0 5.4 52 78.3 70.8 7.5

TABLE 2-3 Anti-HBsAg GMC (mIU/mL) + 95% CI in mITT Subjects with ChronicKidney Disease HEPLISA V ENGERIX-B Week GMC N = 247 GMC N = 260 H/ERatio 4  0.4 0.3 1.58 (0.3, 0.5) (0.2, 0.3) (1.13, 2.20) 8  8.1 0.9 8.84 (5.6, 11.7) (0.7, 1.3)  (5.45, 14.34) 12 16.5 7.2 2.30 (11.7, 23.4) (5.0, 10.4) (1.28, 3.81) 18 33.0 12.0  2.75 (23.4, 46.6)  (8.4, 17.2)(1.67, 4.51) 24 44.3 15.1P11 2.94 (31.6, 62.3) (10.6, 21.5) (1.80, 4.81)28 588.8  156.5  3.76 (386.9, 896) (103.6, 236.3) (2.09, 6.77)

TABLE 2-4 Anti-HBsAg GMC (mIU/mL) + 95% CI in mITT Subjects with ChronicKidney Disease without Diabetes HEPLISA V ENGERIX-B Week GMC GMC H/ERatio 0  0.2  0.2 1.03 (0.2, 0.2) (0.2, 0.2) (0.91, 1.17) 4  0.3  0.31.17 (0.2, 0.5) (0.2, 0.4) (0.68, 2.01) 8  20.6  1.4 =14.66    (11.6,36.6) (0.8, 2.5)  (6.53, 32.90) 12  33.7  12.1 2.79 (19.6, 57.8)  (6.6,22.0) (1.23, 6.32) 18  62.8  24.3 2.58  (36.1, 109.3) (14.0, 42.3)(1.18, 5.66) 24  80.0  27.2 2.94  (45.4, 140.9) (15.8, 47.0) (1.34,6.45) 28 999.0 280.8 3.56  (466.3, 2140.1) (153.1, 515.2) (1.37, 9.24)36 558.7 197.1 2.83  (242.0, 1290.1) (105.7, 367.5) (1.03, 7.79) 44313.6 170.0 1.84 (112.8, 871.8)  (84.9, 340.5) (0.57, 5.98) 52 273.1113.8 2.40  (98.6, 756.4)  (50.4, 256.7) (0.67, 8.54)

TABLE 2-5 Anti-HBsAg GMC (mIU/mL) + 95% CI in mITT Subjects with ChronicKidney Disease with Type II Diabetes HEPLISA V ENGERIX-B Week GMC GMCH/E Ratio 0  0.2 0.2 1.03 (0.2, 0.2) (0.2, 0.2) (0.93, 1.14) 4  0.4 0.21.87 (0.3, 0.6) (0.2, 0.3) (1.23, 2.85) 8  5.3 0.7 7.49 (3.4, 8.3) (0.5,1.0)  (4.13, 13.57) 12  11.8 5.2 2.27  (7.6, 18.3) (3.3, 8.3) 18  24.27.8 3.1  (15.7, 37.2)  (4.9, 12.4) (1.66, 5.81) 24  33.4 10.4  3.20(22.0, 50.9)  (6.6, 16.6) (1.72, 5.95) 28 456.8 109.3  4.18 (276.1,755.7)  (63.1, 189.1) 36 186.7 71.5  2.61 (108.7, 320.6)  (39.6, 129.3)(1.18, 5.79) 44 111.1 44.0  2.53  (63.7, 194.0) (22.7, 85.2) (1.08,5.93) 52 104.9 30.0  3.50  (51.9, 212.1) (14.0, 64.4) (1.25, 9.76)

TABLE 2-6 Anti-HBsAg ≧100 mIU/mL Rates in mITT Subjects with ChronicKidney Disease without Diabetes HEPLISA V ENGERIX-B % Δ Week % SPR Rate% SPR Rate H − E 0 0 0 0 4 1.3 4.0 −2.7 8 34.2 9.2 25.0 12 36.8 24.212.6 18 46.8 31.9 14.8 24 56.6 35.1 21.5 28 80.8 67.4 13.4 36 78.9 63.215.8 44 77.5 64.8 12.7 52 78.6 60.0 18.6

TABLE 2-7 Anti-HBsAg ≧100 mIU/mL Rates in mITT Subjects with ChronicKidney Disease with Type II Diabetes HEPLISA V ENGERIX-B % Δ Week % SPRRate % SPR Rate H − E 0 0 0 0 4 4.8 1.9 2.9 8 12.7 5.7 7.0 12 22.8 20.91.9 18 34.6 22.2 12.4 24 39.6 24.3 15.3 28 70.4 60.7 9.7 36 64.6 55.39.3 44 60.8 52.8 8.0 52 60.0 46.2 14.7

In a Phase 3 trial of adults 18-75 years of age with chronic kidneydisease, three doses of the investigational vaccine HEPLISAVdemonstrated both non-inferiority and superiority to eight doses of thelicensed vaccine ENGERIX-B as determined by measuring seroprotection atweek 28, meeting the primary immunogenicity objective. Similarly, in asubset of chronic kidney disease patients with diabetes, HEPLISAVdemonstrated both non-inferiority and superiority to ENGERIX-B. The twovaccines have similar safety profiles as shown in Table 2-8.

TABLE 2-8 Adverse Experience (AE) Summary HEPLISA V ENGERIX-B Subjectswith N = 254 N = 262 One or more AE 76% 75% Related AE  8%  9%Post-injection reactions 47% 51% systemic 34% 35% pain 28% 34% Severe AE23% 26% Death 2.8%  1.1% 

Tables 2-1 and 2-2 demonstrate that the seroprotection rates in mITTsubjects with chronic kidney disease are significantly higher from weeks8-24 for the subjects treated with HEPLISAV compared to ENGERIX-B.Tables 2-3 through 2-5 show that anti-HBsAg GMC in mITT subjects withchronic kidney disease are significantly higher from weeks 8-52 for thesubjects treated with HEPLISAV compared to ENGERIX-B. Tables 2-6 and 2-7demonstrate that anti-HBsAg ≧100 mIU/mL rates in mITT subjects withchronic kidney disease are significantly higher from weeks 8-52 for thesubjects treated with HEPLISAV compared to ENGERIX-B. Higher antibodylevels in individuals and higher GMCs in the population provide longerlasting protection indicating that fewer and less frequent HEPLISAVboosters are required to maintain seroprotection.

Given the hypo-responsiveness of patients with chronic kidney disease tolicensed hepatitis B vaccines and the increased risk of infection indialysis patients, the availability of a vaccine that provides anearlier response, is more effective using fewer doses, and is moredurable in these patients promises to contribute substantially toprevention of HBV infections.

Although the foregoing disclosure has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications may be practiced. Therefore,descriptions and examples should not be construed as limiting the scopeof the disclosure.

1. A method for eliciting a seroprotective immune response againsthepatitis B virus (HBV) in a diabetic human subject, comprising:administering to a diabetic human subject a first and a second dose ofan immunogenic composition on two separate occasions, wherein saidimmunogenic composition comprises a hepatitis B surface antigen (HBsAg)and an immunostimulatory sequence (ISS) of from 8 to 50 nucleotides inlength comprising an unmethylated cytosine-phosphate-guanosine (CpG)motif, wherein said HBsAg and said ISS are present in said immunogeniccomposition in amounts effective to elicit a seroprotective immuneresponse comprising an anti-HBsAg concentration of at least 10 mIU/ml inthe subject by two months after said second dose.
 2. The method of claim1, wherein the ISS comprises the nucleotide sequence 5′-TCG-3′.
 3. Themethod of claim 1, wherein the ISS comprises the nucleotide sequence‘5-CGTTCG-3’.
 4. The method of claim 1, wherein said ISS comprises thenucleotide sequence of SEQ ID NO:1.
 5. The method of claim 1, whereinsaid ISS comprises a phosphate backbone modification.
 6. The method ofclaim 1, wherein said ISS comprises a phosphorothioate backbonemodification.
 7. The method of claim 1, wherein said ISS issingle-stranded.
 8. The method of claim 1, wherein said immunogeniccomposition comprises about 20 μg of said HBsAg.
 9. The method of claim1, wherein said immunogenic composition comprises about 3000 μg of saidISS.
 10. The method of claim 1, wherein said HBsAg is a recombinantHBsAg produced in yeast.
 11. The method of claim 1, wherein saidseroprotective immune response comprises an anti-HBsAg concentration ofat least 10 mIU/mL in the subject at one month after said second dose.12. The method of claim 11, wherein said anti-HBsAg concentration is atleast 15, 20, or 25 mIU/mL.
 13. The method of claim 1, wherein saidseroprotective immune response comprises an anti-HBsAg concentration ofat least 10 mIU/mL in the subject at six months after said second dose.14. The method of claim 13, wherein said anti-HBsAg concentration is atleast 20, 30, 40 or 50 mIU/mL.
 15. The method of claim 1, wherein thediabetic human subject has type II diabetes.
 16. The method of claim 15,wherein the subject is taking one or both of an oral hypoglycemic andinsulin, at the onset of said administering.
 17. The method of claim 16,wherein the oral hypoglycemic comprises one or more of the groupconsisting of a biguanide, a sulfonylurea, a nonsulfonylureasecretagogue, an alpha glucosidase inhibitor, and a thiazolidinedione.18. The method of claim 1, wherein the diabetic human subject furtherhas chronic kidney disease.
 19. The method of claim 1, wherein said ISSis double-stranded.
 20. A method for eliciting an immune responseagainst hepatitis B virus (HBV) in a human subject who is a member of ahypo-responsive population, comprising: administering to the humansubject a first and a second dose of an immunogenic composition on twoseparate occasions, wherein the immunogenic composition comprises ahepatitis B surface antigen (HBsAg), and an immunostimulatory sequence(ISS) of from 8 to 50 nucleotides in length comprising an unmethylatedcytosine-phosphate-guanosine (CpG) motif, wherein the HBsAg and the ISSare present in the immunogenic composition in amounts effective toelicit an immune response comprising an anti-HBsAg concentration of atleast 5 mUI/mL in the human subject by two months after the second dose.21. The method of claim 20, wherein said hypo-responsive populationcomprises one or more of older adults, males, obese individuals,smokers, diabetics, and patients with chronic kidney disease.
 22. Themethod of claim 20, wherein said immune response is a seroprotectiveimmune response comprising an anti-HBsAg concentration of at least 10mUI/mL in the human subject by two months after the second dose.
 23. Amethod for eliciting a seroprotective immune response against hepatitisB virus (HBV) in a human subject with chronic kidney disease accordingto an abbreviated vaccination schedule, comprising: administering to thehuman subject with chronic kidney disease an immunogenic compositionaccording to an abbreviated vaccination schedule, wherein theabbreviated vaccination schedule comprises administering a first, secondand third dose of the immunogenic composition on three separateoccasions, wherein the immunogenic composition comprises a hepatitis Bsurface antigen (HBsAg), and an immunostimulatory sequence (ISS) of from8 to 50 nucleotides in length comprising an unmethylatedcytosine-phosphate-guanosine (CpG) motif, wherein the HBsAg and the ISSare present in the immunogenic composition in amounts effective toelicit a seroprotective immune response comprising an anti-HBsAgconcentration of at least 10 mUI/mL in the human subject by one monthafter the third dose.
 24. A method for eliciting a durable immuneresponse against hepatitis B virus (HBV) in a human subject with chronickidney disease according to an abbreviated vaccination schedule,comprising: administering to the human subject an immunogeniccomposition according to an abbreviated vaccination schedule, whereinthe abbreviated vaccination schedule comprises administering a first,second and third dose of the immunogenic composition on three separateoccasions, wherein the immunogenic composition comprises a hepatitis Bsurface antigen (HBsAg), and an immunostimulatory sequence (ISS) of from8 to 50 nucleotides in length comprising an unmethylatedcytosine-phosphate-guanosine (CpG) motif, wherein the HBsAg and the ISSare present in the immunogenic composition in amounts effective toelicit a durable immune response comprising an anti-HBsAg concentrationof at least 100 in the human subject 6 months or later after the thirddose.
 25. The method of claim 24, wherein said durable immune responsecomprises an anti-HBsAg concentration of at least 100 mIU/mL in thehuman subject at one month after said third dose.